Transgenic plants modified for reduced cadmium transport, derivative products, and related methods

ABSTRACT

Various embodiments are directed to transgenic plants, including transgenic tobacco plants and derivative seeds, genetically modified to impede the transport of Cadmium (Cd) from the root system to aerial portions of transgenic plants by reducing the expression levels of HMA-related transporters. Various embodiments are directed to transgenic tobacco plants genetically modified to stably express a RNAi construct encoding RNAi polynucleotides that enable the degradation of endogenous NtHMA RNA variants. Reduced expression of NtHMA transporters in transgenic plants results in substantially reduced content of Cadmium (Cd) in the leaf lamina. Various consumable products that are substantially free or substantially reduced in Cd content can be produced by incorporating leaves derived from transgenic tobacco plants modified to reduce the expression of NtHMA transporters.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Divisional Application of U.S. patent applicationSer. No. 13/770,702, filed on Feb. 19, 2013, which is a DivisionalApplication of U.S. patent application Ser. No. 12/333,681, filed onDec. 12, 2008, which issued on Feb. 26, 2013 as U.S. Pat. No. 8,383,889,which claims priority under 35 U.S.C. §119 to U.S. ProvisionalApplication Ser. No. 60/996,982, filed on Dec. 13, 2007, and the contentof each is hereby expressly incorporated by reference.

SEQUENCE LISTING

This application hereby incorporates by reference the text file filedelectronically herewith having the name “1021238-000933 sequencelisting.txt” created on Nov. 20, 2008 with a file size of 125,091 bytes.

TECHNICAL FIELD

Compositions, expression vectors, polynucleotides, polypeptides,transgenic plants, transgenic cell lines, and transgenic seeds, andmethods for making and using these embodiments to produce various plantsthat can reduce the transport of heavy metals into aerial portions.

BACKGROUND

Plants obtain essential heavy metals, such as Zn, Ni, and Cu, byabsorbing metal ion substrates from their environment by varioustransport mechanisms mediated by transmembrane transporters expressed onthe surface of root cells and other vascular tissues. Transportersclassified as P-type ATPases, such as P1B-type ATPases, are transportersthat translocate positively charged substrates across plasma membranesby utilizing energy liberated from exergonic ATP hydrolysis reactions.P1B-type ATPases are also referred to as heavy metal ATP-ases (“HMAs”)or CPx-type ATPases. HMAs have been grouped by substrate specificityinto two subclasses, the Cu/Ag and Zn/Co/Cd/Pb groups. The firstP1B-type ATPase to be characterized in plants is AtHMA4, cloned fromArabidopsis. Substrate selectivity by HMAs is not strictly limited tothe transport of essential metals in that several non-essential metalscan be recognized indiscriminantly as substrates, resulting in theaccumulation of many non-essential metals, such as Cd, Pb, As, and Hg.

SUMMARY

Various embodiments are directed to compositions and methods forproducing transgenic plants, including transgenic tobacco plants,genetically modified to impede Cadmium (Cd) transport from the rootsystem to the leaf lamina by reducing the expression levels oftransporters of the HMA family. A HMA homologue (“NtHMA”) has beenidentified in tobacco, which can be utilized for constructing variousRNAi constructs, encoding NtHMA RNAi polynucleotides of interest thatcan facilitate the degradation of endogenous NtHMA RNA transcripts.Transgenic plants that can express NtHMA RNAi polynucleotides accordingto this disclosure can be utilized for reducing steady-state levels ofNtHMA RNA transcripts, and consequently, for reducing the number offunctionally active NtHMA transporters available for transporting metalsacross cellular membranes.

Various embodiments are directed to recombinant expression vectorscomprising various NtHMA RNAi constructs, transgenic plants and seedsgenetically modified to exogenously express NtHMA RNAi polynucleotides,cell lines derived from transgenic plants and seeds, and consumableproducts incorporating leaves derived from transgenic plants producedaccording to the disclosed methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic of a NtHMA genomic clone comprising 11 exons.FIG. 1B provides a list of nucleotide positions mapped to each exonwithin the isolated NtHMA genomic clone (“Table 1”).

FIG. 2A illustrates an exemplary subcloning strategy for constructing aNtHMA RNAi expression vector that enables the constitutive expression ofNtHMA RNAi polynucleotides of interest, as described in Example 2.

FIG. 2B illustrates a hypothetical double-stranded RNA duplex formed (as“stem-loop-stem” structure) from intra-molecular, base-pair interactionswithin NtHMA RNAi polynucleotide produced as a product transcribed froman exemplary NtHMA RNAi construct.

FIG. 3A shows an exemplary RNAi sequence, NtHMA (660-915), for producingNtHMA RNAi polynucleotides of interest, as described in Example 2.

FIGS. 3B-3D show Cd reduction in leaf lamina of multiple firstgeneration (T0) transgenic lines, representing three varieties, thathave been genetically modified to express NtHMA RNAi polynucleotides(660-915), as described in Example 5.

FIG. 4A shows an exemplary RNAi sequence, NtHMA (1382-1584), forproducing NtHMA RNAi polynucleotides of interest, as described inExample 3.

FIGS. 4B-4D show Cd reduction in leaf lamina of multiple firstgeneration (T0) transgenic lines, representing three varieties, thathave been genetically modified to express NtHMA RNAi polynucleotides(1382-1584), as described in Example 5.

FIGS. 5A-C show normalized NtHMA RNA transcript levels in various firstgeneration (T0) transgenic lines that have been genetically modified toexpress NtHMA RNAi polynucleotides of interest, as determined byquantitative realtime PCR analysis of leaf lamina extracts, as describedin Example 6.

FIG. 6 shows the distribution of Cd and Zn between the leaf lamina andthe root of various first generation (T0) transgenic lines that havebeen genetically modified to express NtHMA RNAi polynucleotides ofinterest, as presented in Table 2 and described in Example 7.

FIG. 7 shows Cd distribution among the bark, leaf lamina, pith, and roottissues of various first generation (T0) transgenic lines that have beengenetically modified to express NtHMA RNAi polynucleotides of interest,as presented in Table 3 and described in Example 8.

FIG. 8 shows Cd distribution between the leaf lamina and the root ofvarious second generation (T1) transgenic lines that have beengenetically modified to express NtHMA RNAi polynucleotides of interest,as described in Example 9.

DETAILED DESCRIPTION I. Isolation of Tobacco NtHMA Genes and GeneProducts

FIG. 1A is a schematic of a NtHMA genomic clone comprising 11 exonsencoding a heavy metal transporter related to the HMA family oftransporters. Example 1 further describes the identification of theNtHMA genomic clone (_HO-18-2) and 4 NtHMA cDNA clones. FIG. 1B.provides nucleotide positions corresponding to exon and intronsubregions mapped within the NtHMA genomic clone (_HO-18-2).

A. NtHMA Polynucleotides

The term “polynucleotide” refers to a polymer of nucleotides comprisingat least 10 bases in length. The polynucleotides may be DNA, RNA or aDNA/RNA hybrid, comprising ribonucleotides, deoxyribonucleotides,combinations of deoxyribo- and ribo-nucleotides, and combinations ofbases and/or modifications, including uracil, adenine, thymine,cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, andisoguanine. The term includes single and double-stranded forms of DNA orRNA. The term “DNA” includes genomic DNAs, cDNAs, chemically-synthesizedDNAs, PCR-amplified DNAs, and combinations/equivalents thereof. The term“isolated polynucleotide” refers to a polynucleotide not contiguous withany genome of origin, or separated from a native context. The termincludes any recombinant polynucleotide molecule such as NtHMA RNAiconstructs, NtHMA RNAi expression vectors, NtHMA genomic clones, andfragments and variants thereof.

As shown in FIG. 1A, the NtHMA genomic clone, designated as SEQ ID NO:1,comprises: intron 1 (SEQ ID NO:4), exon 1 (SEQ ID NO:5), intron 2 (SEQID NO:6), exon 2 (SEQ ID NO:7), intron 3 (SEQ ID NO:8), exon 3 (SEQ IDNO:9), intron 4 (SEQ ID NO:10), exon 4 (SEQ ID NO:11), intron 5 (SEQ IDNO:12), exon 5 (SEQ ID NO:13), intron 6 (SEQ ID NO:14), exon 6 (SEQ IDNO:15), intron 7 (SEQ ID NO:16), exon 7 (SEQ ID NO:17), intron 8 (SEQ IDNO:18), exon 8 (SEQ ID NO:19), intron 9 (SEQ ID NO:20), exon 9 (SEQ IDNO:21), intron 10 (SEQ ID NO:22), exon 10 (SEQ ID NO:23), intron 11 (SEQID NO:24, exon 11 (SEQ ID NO:25), and intron 12 (SEQ ID NO:26). Variousembodiments are directed to isolated polynucleotides representinggenomic fragments isolated at the NtHMA locus, comprising SEQ ID NO:1,fragments of SEQ ID NO:1, or variants thereof. Various embodiments aredirected to isolated NtHMA polynucleotide variants comprising at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%sequence identity to SEQ ID NO:1, or fragments of SEQ ID NO:1.

Various embodiments are directed to isolated polynucleotides havingsequences that complements that of NtHMA polynucleotide variantscomprising at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:1, or fragments ofSEQ ID NO:1. Various embodiments are directed to isolatedpolynucleotides that can specifically hybridize, under moderate tohighly stringent conditions, to polynucleotides comprising SEQ ID NO:1,or fragments of SEQ ID NO:1.

Various embodiments are directed to isolated polynucleotides of NtHMAcDNA (Clone P6663), comprising SEQ ID NO:3, fragments of SEQ ID NO:3, orvariants thereof. Various embodiments are directed to isolated NtHMApolynucleotide variants comprising at least 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:3, orfragments of SEQ ID NO:3. Various embodiments are directed to isolatedNtHMA polyribonucleotide variants comprising at least 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:3,or fragments of SEQ ID NO:3, and in which Ts have been substituted withUs (e.g., RNAs). Various embodiments are directed to isolatedpolynucleotides that can specifically hybridize, under moderate tohighly stringent conditions, to polynucleotides comprising SEQ ID NO:3,or fragments of SEQ ID NO:3. Various embodiments are directed toisolated polynucleotides having a sequence that complements that ofNtHMA polynucleotide variants comprising at least 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:3, orfragments of SEQ ID NO:3.

Various embodiments are directed to isolated polynucleotides of NtHMAcDNA (Clone P6643), comprising SEQ ID NO:47, fragments of SEQ ID NO:47,or variants thereof. Various embodiments are directed to isolated NtHMApolynucleotide variants comprising at least 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:47, fragmentsof SEQ ID NO:47. Various embodiments are directed to isolated NtHMApolyribonucleotide variants comprising at least 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:47, fragmentsof SEQ ID NO:47, and in which Ts have been substituted with Us (e.g.,RNAs). Various embodiments are directed to isolated polynucleotides thatcan specifically hybridize, under moderate to highly stringentconditions, to polynucleotides comprising SEQ ID NO:47, fragments of SEQID NO:47. Various embodiments are directed to isolated polynucleotideshaving a sequence that complements that of NtHMA polynucleotide variantscomprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%sequence identity to SEQ ID NO:47, fragments of SEQ ID NO:47.

Various embodiments are directed to biopolymers that are homologous toNtHMA polynucleotides and NtHMA polypeptides (“NtHMA homologues”), whichcan be identified from different plant species. For example, NtHMAhomologues can be experimentally isolated by screening suitable nucleicacid libraries derived from different plant species of interest.Alternatively, NtHMA homologues may be identified by screening genomedatabases containing sequences from one or more species utilizing asequence derived from NtHMA polynucleotides and/or NtHMA polypeptides.Such genomic databases are readily available for a number of species(e.g., on the world wide web (www) at tigr.org/tdb; genetics.wisc.edu;stanford.edu/.about.ball; hiv-web.lan1.gov; ncbi.nlm.nig.gov; ebi.ac.uk;and pasteur.fr/other/biology). For example, degenerate oligonucleotidesequences can be obtained by “back-translation” from NtHMA polypeptidefragments. NtHMA polynucleotides can be utilized as probes or primers toidentify/amplify related sequences, or to obtain full-length sequencesfor related NtHMAs by PCR, for example, or by other well-knowntechniques (e.g., see PCR Protocols: A Guide to Methods andApplications, Innis et. al., eds., Academic Press, Inc. (1990)).

B. NtHMA Polypeptides

The term “NtHMA polypeptide” refers to a polypeptide comprising an aminoacid sequence designated as SEQ ID NO:2; polypeptides having substantialhomology (i.e., sequence similarity) or substantial identity to SEQ IDNO:2; fragments of SEQ ID NO:2; and variants thereof. The NtHMApolypeptides include sequences having sufficient or substantial degreeof identity or similarity to SEQ ID NO:2, and that can function bytransporting heavy metals across cell membranes.

NtHMA polypeptides include variants produced by introducing any type ofalterations (e.g., insertions, deletions, or substitutions of aminoacids; changes in glycosylation states; changes that affect refolding orisomerizations, three-dimensional structures, or self-associationstates), which can be deliberately engineered or isolated naturally.NtHMA polypeptides may be in linear form or cyclized using known methods(e.g., H. U. Saragovi, et al., Bio/Technology 10, 773 (1992); and R. S.McDowell, et al., J. Amer. Chem. Soc. 114:9245 (1992), both incorporatedherein by reference). NtHMA polypeptides comprise at least 8 to 10, atleast 20, at least 30, or at least 40 contiguous amino acids.

Various embodiments are directed to isolated NtHMA polypeptides encodedby polynucleotide sequence, SEQ ID NO:1, comprising SEQ ID NO:2,fragments of SEQ ID NO:2, or variants thereof. Various embodiments aredirected to isolated NtHMA polypeptide variants comprising at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQID NO:2, or fragments of SEQ ID NO:2.

Various embodiments are directed to isolated NtHMA polypeptides (CloneP6663), comprising SEQ ID NO:2, fragments of SEQ ID NO:2, or variantsthereof. Various embodiments are directed to isolated NtHMA polypeptidevariants comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, and 99% sequence identity to SEQ ID NO:2, or fragments of SEQ IDNO:2.

Various embodiments are directed to isolated NtHMA polypeptides (CloneP6643), comprising SEQ ID NO:49, fragments of SEQ ID NO:49, or variantsthereof. Various embodiments are directed to isolated NtHMA polypeptidevariants comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, and 99% sequence identity to SEQ ID NO:49, or fragments of SEQ IDNO:49.

II. Compositions and Related Methods for Reducing NtHMA Gene Expressionand/or NtHMA-Mediated Transporter Activity

Suitable antagonistic compositions that can down-regulate the expressionand/or the activity of NtHMA and NtHMA variants includesequence-specific polynucleotides that can interfere with thetranscription of one or more endogenous NtHMA gene(s); sequence-specificpolynucleotides that can interfere with the translation of NtHMA RNAtranscripts (e.g., dsRNAs, siRNAs, ribozymes); sequence-specificpolypeptides that can interfere with the protein stability of NtHMA, theenzymatic activity of NtHMA, and/or the binding activity of NtHMA withrespect to substrates and/or regulatory proteins; antibodies thatexhibit specificity for NtHMA; and small molecule compounds that caninterfere with the protein stability of NtHMA, the enzymatic activity ofNtHMA, and/or the binding activity of NtHMA. An effective antagonist canreduce heavy metal (e.g., Cd) transport into leaf lamina structures byat least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, or 95%.

A. Definitions

Throughout this disclosure and the appended claims, the terms “a” and“the” function as singular and plural referents unless the contextclearly dictates otherwise. Thus, for example, a reference to “an RNAipolynucleotide” includes a plurality of such RNAi polynucleotides, and areference to “the plant” includes reference to one or more of suchplants.

The term “orientation” refers to a particular order in the placement ofa polynucleotide relative to the position of a reference polynucleotide.A linear DNA has two possible orientations: the 5′-to-3′ direction andthe 3′-to-5′ direction. For example, if a reference sequence ispositioned in the 5′-to-3′ direction, and if a second sequence ispositioned in the 5′-to-3′ direction within the same polynucleotidemolecule/strand, then the reference sequence and the second sequence areorientated in the same direction, or have the same orientation.Typically, a promoter sequence and a gene of interest under theregulation of the given promoter are positioned in the same orientation.However, with respect to the reference sequence positioned in the5′-to-3′ direction, if a second sequence is positioned in the 3′-to-5′direction within the same polynucleotide molecule/strand, then thereference sequence and the second sequence are orientated in anti-sensedirection, or have anti-sense orientation. Two sequences havinganti-sense orientations with respect to each other can be alternativelydescribed as having the same orientation, if the reference sequence(5′-to-3′ direction) and the reverse complementary sequence of thereference sequence (reference sequence positioned in the 5′-to-3′) arepositioned within the same polynucleotide molecule/strand.

The term “NtHMA RNAi expression vector” refers to a nucleic acid vehiclethat comprises a combination of DNA components for enabling thetransport and the expression of NtHMA RNAi constructs. Suitableexpression vectors include episomes capable of extra-chromosomalreplication such as circular, double-stranded DNA plasmids; linearizeddouble-stranded DNA plasmids; and other functionally equivalentexpression vectors of any origin. A suitable NtHMA RNAi expressionvector comprises at least a promoter positioned upstream andoperably-linked to a NtHMA RNAi construct, as defined below.

The term “NtHMA RNAi construct” refers to a double-stranded, recombinantDNA fragment that encodes “NtHMA RNAi polynucleotides” having RNAinterference activity. A NtHMA RNAi construct comprises a “templatestrand” base-paired with a complementary “sense or coding strand.” Agiven NtHMA RNAi construct can be inserted into a NtHMA RNAi expressionvector in two possible orientations, either in the same (or sense)orientation or in the reverse (or anti-sense) orientation with respectto the orientation of a promoter positioned within a NtHMA RNAiexpression vector.

The term “NtHMA RNAi polynucleotides” can target NtHMA RNA for enzymaticdegradation, involving the formation of smaller fragments of NtHMA RNAipolynucleotides (“siRNAs”) that can bind to multiple complementarysequences within the target NtHMA RNA. Expression levels of one or moreNtHMA gene(s) can be reduced by the RNA interference activity of NtHMARNAi polynucleotides.

The term “template strand” refers to the strand comprising a sequencethat complements that of the “sense or coding strand” of a DNA duplex,such as NtHMA genomic fragment, NtHMA cDNA, or NtHMA RNAi construct, orany DNA fragment comprising a nucleic acid sequence that can betranscribed by RNA polymerase. During transcription, RNA polymerase cantranslocate along the template strand in the 3′-to-5′ direction duringnacent RNA synthesis.

The terms “sense strand” or “coding strand” refer to the strandcomprising a sequence that complements that of the template strand in aDNA duplex. For example, the sequence of the sense strand (“sensesequence”) for the identified NtHMA genomic clone is designated as SEQID NO:1. For example, the sense sequence for NtHMA cDNA, identified asclone P6663, is designated as SEQ ID NO:3. For example, the sensesequence for NtHMA cDNA, identified as clone P6643, is designated as SEQID NO:46. For example, if the sense strand comprises a hypotheticalsequence 5′-TAATCCGGT-3′ (SEQ ID NO:50), then the substantiallyidentical corresponding sequence within a hypothetical target mRNA is5′-UAAUCCGGU-3′ (SEQ ID NO:51).

The term “reverse complementary sequence” refers to the sequence thatcomplements the “sense sequence” of interest (e.g., exon sequence)positioned within the same strand, in the same orientation with respectto the sense sequence. For example, if a strand comprises a hypotheticalsequence 5′-TAATCCGGT-3′ (SEQ ID NO:52), then the reverse complementarysequence 5′-ACCGGATTA-3′ (SEQ ID NO:53) may be operably-linked to thesense sequence, separated by a spacer sequence.

The terms “NtHMA RNA transcript” or “NtHMA RNA,” in the context of RNAinterference, refer to polyribonucleic acid molecules produced within ahost plant cell of interest, resulting from the transcription ofendogenous genes of the HMA family, including the isolated NtHMA gene(SEQ ID NO:1). Thus, these terms include any RNA species or RNA variantsproduced as transcriptional products from HMA-related genes that may bedistinct from the isolated NtHMA gene (SEQ ID NO:1) but havingsufficient similarity at structural and/or functional levels to beclassified within the same family. For example, if a host plant cellselected for genetic modification according to the disclosed methods istobacco, then target NtHMA RNA transcripts include: (1) pre-mRNAs andmRNAs produced from the transcription of the isolated NtHMA gene (SEQ IDNO:1); (2) pre-mRNAs and mRNAs produced from the transcription of anygenes having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, and 99% sequence identity to the sequence of the isolatedNtHMA gene (SEQ ID NO:1) (i.e. other distinct genes substantiallyidentical to the identified NtHMA gene and encoding related isoforms ofHMA transporters); and (3) pre-mRNAs and mRNAs produced from thetranscription of alleles of the NtHMA gene (SEQ ID NO:1). The NtHMA RNAtranscripts include RNA variants produced as a result of alternative RNAsplicing reactions of heteronuclear RNAs (“hnRNAs”) of a particularNtHMA gene, mRNA variants resulting from such alternative RNA splicingreactions, and any intermediate RNA variants.

The terms “homology” or “identity” or “similarity” refer to the degreeof sequence similarity between two polypeptides or between two nucleicacid molecules compared by sequence alignment. The degree of homologybetween two discrete nucleic acid sequences being compared is a functionof the number of identical, or matching, nucleotides at comparablepositions. The percent identity may be determined by visual inspectionand mathematical calculation. Alternatively, the percent identity of twonucleic acid sequences can be determined by comparing sequenceinformation using the GAP computer program, version 6.0 described byDevereux et al. (Nucl. Acids Res. 12:387, 1984) and available from theUniversity of Wisconsin Genetics Computer Group (UWGCG). Typical defaultparameters for the GAP program include: (1) a unary comparison matrix(containing a value of 1 for identities and 0 for non-identities) fornucleotides, and the weighted comparison matrix of Gribskov and Burgess,Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff,eds., Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for eachgap and an additional 0.10 penalty for each symbol in each gap; and (3)no penalty for end gaps. Various programs known to persons skilled inthe art of sequence comparison can be alternatively utilized.

The term “upstream” refers to a relative direction/position with respectto a reference element along a linear polynucleotide sequence, whichindicates a direction/position towards the 5′ end of the polynucleotidesequence. “Upstream” may be used interchangeably with the “5′ end of areference element.”

The term “operably-linked” refers to the joining of distinct DNAelements, fragments, or sequences to produce a functionaltranscriptional unit or a functional expression vector.

The term “promoter” refers to a nucleic acid element/sequence, typicallypositioned upstream and operably-linked to a double-stranded DNAfragment, such as a NtHMA RNAi construct. For example, a suitablepromoter enables the transcriptional activation of a NtHMA RNAiconstruct by recruiting the transcriptional complex, including the RNApolymerase and various factors, to initiate RNA synthesis. “Promoters”can be derived entirely from regions proximate to a native gene ofinterest, or can be composed of different elements derived fromdifferent native promoters and/or synthetic DNA segments. Suitablepromoters include tissue-specific promoters recognized bytissue-specific factors present in different tissues or cell types(e.g., root-specific promoters, shoot-specific promoters, xylem-specificpromoters), or present during different developmental stages, or presentin response to different environmental conditions. Suitable promotersinclude constitutive promoters that can be activated in most cell typeswithout requiring specific inducers. Examples of suitable promoters forcontrolling NtHMA RNAi polypeptide production include the cauliflowermosaic virus 35S (CaMV/35S), SSU, OCS, lib4, usp, STLS1, B33, nos orubiquitin- or phaseolin-promoters. Persons skilled in the art arecapable of generating multiple variations of recombinant promoters, asdescribed in a number of references, such as Okamuro and Goldberg,Biochemistry of Plants, Vol. 15:pp 1-82 (1989).

Tissue-specific promoters are transcriptional control elements that areonly active in particular cells or tissues at specific times duringplant development, such as in vegetative tissues or reproductivetissues. Tissue-specific expression can be advantageous, for example,when the expression of polynucleotides in certain tissues is preferred.Examples of tissue-specific promoters under developmental controlinclude promoters that can initiate transcription only (or primarilyonly) in certain tissues, such as vegetative tissues, e.g., roots orleaves, or reproductive tissues, such as fruit, ovules, seeds, pollen,pistols, flowers, or any embryonic tissue. Reproductive tissue-specificpromoters may be, e.g., anther-specific, ovule-specific,embryo-specific, endosperm-specific, integument-specific, seed and seedcoat-specific, pollen-specific, petal-specific, sepal-specific, orcombinations thereof.

Suitable leaf-specific promoters include pyruvate, orthophosphatedikinase (PPDK) promoter from C4 plant (maize), cab-m1 Ca+2 promoterfrom maize, the Arabidopsis thaliana myb-related gene promoter (Atmyb5),the ribulose biphosphate carboxylase (RBCS) promoters (e.g., the tomatoRBCS 1, RBCS2 and RBCS3A genes expressed in leaves and light-grownseedlings, RBCS1 and RBCS2 expressed in developing tomato fruits, and/orribulose bisphosphate carboxylase promoter expressed almost exclusivelyin mesophyll cells in leaf blades and leaf sheaths at high levels).

Suitable senescence-specific promoters include a tomato promoter activeduring fruit ripening, senescence and abscission of leaves, a maizepromoter of gene encoding a cysteine protease. Suitable anther-specificpromoters can be used. Such promoters are known in the art or can bediscovered by known techniques; see, e.g., Bhalla and Singh (1999)Molecular control of male fertility in Brassica, Proc. 10th AnnualRapeseed Congress, Canberra, Australia; van Tunen et al. (1990) Pollen-and anther-specific chi promoters from petunia: tandem promoterregulation of the chiA gene. Plant Cell 2:393-40; Jeon et al. (1999)Isolation and characterization of an anther-specific gene, RA8, fromrice (Oryza sativa L). Plant Molecular Biology 39:35-44; and Twell etal. (1993) Activation and developmental regulation of an Arabidopsisanther-specific promoter in microspores and pollen of Nicotiana tabacum.Sex. Plant Reprod. 6:217-224.

Suitable root-preferred promoters known to persons skilled in the artmay be selected. See, for example, Hire et al. (1992) Plant Mol. Biol.20(2):207-218 (soybean root-specific glutamine synthetase gene); Kellerand Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specific controlelement in the GRP 1.8 gene of French bean); Sanger et al. (1990) PlantMol. Biol. 14(3):433-443 (root-specific promoter of the mannopinesynthase (MAS) gene of Agrobacterium tumefaciens); and Miao et al.(1991) Plant Cell 3(1):11-22 (full-length cDNA clone encoding cytosolicglutamine synthetase (GS), which is expressed in roots and root nodulesof soybean). See also Bogusz et al. (1990) Plant Cell 2(7):633-641,where two root-specific promoters isolated from hemoglobin genes fromthe nitrogen-fixing nonlegume Parasponia andersonii and the relatednon-nitrogen-fixing nonlegume Trema tomentosa are described.

Suitable seed-preferred promoters include both seed-specific promoters(those promoters active during seed development such as promoters ofseed storage proteins) and seed-germinating promoters (those promotersactive during seed germination). See, e.g., Thompson et al. (1989)BioEssays 10: 108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1-phosphatesynthase); mZE40-2, also known as Zm-40 (U.S. Pat. No. 6,403,862); nucic(U.S. Pat. No. 6,407,315); and celA (cellulose synthase) (see WO00/11177). Gama-zein is an endosperm-specific promoter. Glob-1 is anembryo-specific promoter. For dicots, seed-specific promoters include,but are not limited to, bean .beta.-phaseolin, napin,.beta.-conglycinin, soybean lectin, cruciferin, and the like. Formonocots, seed-specific promoters include, but are not limited to, amaize 15 kDa zein promoter, a 22 kDa zein promoter, a 27 kDa zeinpromoter, a g-zein promoter, a 27 kD .gamma.-zein promoter (such asgzw64A promoter, see Genbank Accession #S78780), a waxy promoter, ashrunken 1 promoter, a shrunken 2 promoter, a globulin 1 promoter (seeGenbank Accession # L22344), an Itp2 promoter (Kalla, et al., PlantJournal 6:849-860 (1994); U.S. Pat. No. 5,525,716), cim1 promoter (seeU.S. Pat. No. 6,225,529) maize end1 and end2 promoters (See U.S. Pat.No. 6,528,704 and application Ser. No. 10/310,191, filed Dec. 4, 2002);nuc1 promoter (U.S. Pat. No. 6,407,315); Zm40 promoter (U.S. Pat. No.6,403,862); eep1 and eep2; lec1 (U.S. patent application Ser. No.09/718,754); thioredoxinH promoter (U.S. provisional patent application60/514,123); mlip15 promoter (U.S. Pat. No. 6,479,734); PCNA2 promoter;and the shrunken-2 promoter. (Shaw et al., Plant Phys 98:1214-1216,1992; Zhong Chen et al., PNAS USA 100:3525-3530, 2003).

Examples of inducible promoters include promoters responsive to pathogenattack, anaerobic conditions, elevated temperature, light, drought, coldtemperature, or high salt concentration. Pathogen-inducible promotersinclude those from pathogenesis-related proteins (PR proteins), whichare induced following infection by a pathogen (e.g., PR proteins, SARproteins, beta-1,3-glucanase, chitinase). See, for example, Redolfi etal. (1983) Neth. J. Plant Pathol. 89:245-254; Uknes et al. (1992) PlantCell 4:645-656; and Van Loon (1985) Plant Mol. Virol. 4:111-116. Seealso the application entitled “Inducible Maize Promoters”, U.S. patentapplication Ser. No. 09/257,583, filed Feb. 25, 1999.

In addition to plant promoters, other suitable promoters may be derivedfrom bacterial origin (e.g., the octopine synthase promoter, thenopaline synthase promoter and other promoters derived from Tiplasmids), or may be derived from viral promoters (e.g., 35S and 19S RNApromoters of cauliflower mosaic virus (CaMV), constitutive promoters oftobacco mosaic virus, cauliflower mosaic virus (CaMV) 19S and 35Spromoters, or figwort mosaic virus 35S promoter).

The term “enhancer” refers to a nucleic acid molecule, or a nucleic acidsequence, that can recruit transcriptional regulatory proteins such astranscriptional activators, to enhance transcriptional activation byincreasing promoter activity. Suitable enhancers can be derived fromregions proximate to a native promoter of interest (homologous sources)or can be derived from non-native contexts (heterologous sources) andoperably-linked to any promoter of interest within NtHMA RNAi expressionvectors to enhance the activity and/or the tissue-specificity of apromoter. Some enhancers can operate in any orientation with respect tothe orientation of a transcription unit. For example, enhancers may bepositioned upstream or downstream of a transcriptional unit comprising apromoter and a NtHMA RNAi construct. Persons skilled in the art arecapable of operably-linking enhancers and promoters to optimize thetranscription levels of NtHMA RNAi constructs.

B. RNAi Expression Vectors Comprising NtHMA RNAi Constructs encodingNtHMA RNAi Polynucleotides

RNA Interference (“RNAi”) or RNA silencing is an evolutionarilyconserved process by which specific mRNAs can be targeted for enzymaticdegradation. A double-stranded RNA (dsRNA) must be introduced orproduced by a cell (e.g., dsRNA virus, or NtHMA RNAi polynucleotides) toinitiate the RNAi pathway. The dsRNA can be converted into multiplesiRNA duplexes of 21-23 bp length (“siRNAs”) by RNases III, which aredsRNA-specific endonucleases (“Dicer”). The siRNAs can be subsequentlyrecognized by RNA-induced silencing complexes (“RISC”) that promote theunwinding of siRNA through an ATP-dependent process. The unwoundantisense strand of the siRNA guides the activated RISC to the targetedmRNA (e.g., NtHMA RNA variants) comprising a sequence complementary tothe siRNA anti-sense strand. The targeted mRNA and the anti-sense strandcan form an A-form helix, and the major groove of the A-form helix canbe recognized by the activated RISC. The target mRNA can be cleaved byactivated RISC at a single site defined by the binding site of the5′-end of the siRNA strand. The activated RISC can be recycled tocatalyze another cleavage event.

FIG. 2A illustrates the construction of an exemplary NtHMA RNAiexpression vector. Various embodiments are directed to NtHMA RNAiexpression vectors comprising NtHMA RNAi constructs encoding NtHMA RNAipolynucleotides exhibiting RNA interference activity by reducing theexpression level of NtHMA mRNAs, NtHMA pre-mRNAs, and related NtHMA RNAvariants. The expression vectors comprise a promoter positioned upstreamand operably-linked to a NtHMA RNAi construct, as further defined below.NtHMA RNAi expression vectors comprise a suitable minimal core promoter,a NtHMA RNAi construct of interest, an upstream (5′) regulatory region,a downstream (3′) regulatory region, including transcription terminationand polyadenylation signals, and other sequences known to personsskilled in the art, such as various selection markers.

The NtHMA polynucleotides can be produced in various forms, including asdouble-stranded hairpin-like structures (“dsRNAi”). The NtHMA dsRNAi canbe enzymatically converted to double-stranded NtHMA siRNAs. One of thestrands of the NtHMA siRNA duplex can anneal to a complementary sequencewithin the target NtHMA mRNA and related NtHMA RNA variants. ThesiRNA/mRNA duplexes are recognized by RISC that can cleave NtHMA RNAs atmultiple sites in a sequence-dependent manner, resulting in thedegradation of the target NtHMA mRNA and related NtHMA RNA variants.

FIG. 2B illustrates the formation of a hypothetical double-stranded RNAduplex formed (as “stem-loop-stem” structure) as a product transcribedfrom an exemplary NtHMA RNAi construct. In FIG. 2B, a hypothetical NtHMARNAi construct 10 is shown, comprising 3 double-stranded DNA fragments,such as fragments 1-3. Fragment 1 is positioned upstream andoperably-linked to fragment 2, which is positioned upstream andoperably-linked to fragment 3, for which DNA strands/sequences 4, 6, and8 are liked together in tandem to form strand 11, as shown.Alternatively, a NtHMA RNAi construct comprises “a sense sequence” 5,which is positioned upstream and operably-linked to “a spacer sequence”7, which is positioned upstream and operably-linked to “a reversecomplementary sequence” 9. The strands/sequences 5, 7, and 9 can beliked together in tandem to form strand/sequence 12. Alternatively, aNtHMA RNAi construct comprises “a sense sequence” 8, which is positionedupstream and operably-linked to “a spacer sequence” 6, which ispositioned upstream and operably-linked to “a reverse complementarysequence” 4. The strands/sequences 8, 6, and 4 can be liked together intandem to form strand/sequence 11. Strand 12 is complementary to strand11. Strand 11 is a template strand that can be transcribed into a NtHMARNAi polynucleotide 13. The NtHMA RNAi polynucleotide 13 forms ahair-pin (“stem-loop-stem”) structure, in which the stem 16 is acomplementary region resulting from intra-molecular base-pairinteractions of the NtHMA RNAi polynucleotide 15 and the loop 17represents a non-complementary region encoded by a spacer sequence, suchas strands/sequences 6 or 7.

Any NtHMA RNA polynucleotide of interest can be produced by selecting asuitable sequence composition, loop size, and stem length for producingthe NtHMA hairpin duplex. A suitable range for designing stem lengths ofa hairpin duplex, includes stem lengths of 20-30 nucleotides, 30-50nucleotides, 50-100 nucleotides, 100-150 nucleotides, 150-200nucleotides, 200-300 nucleotides, 300-400 nucleotides, 400-500nucleotides, 500-600 nucleotides, and 600-700 nucleotides. A suitablerange for designing loop lengths of a hairpin duplex, includes looplengths of 4-25 nucleotides, 25-50 nucleotides, or longer if the stemlength of the hair duplex is substantial. In certain context, hairpinstructures with duplexed regions longer than 21 nucleotides may promoteeffective siRNA-directed silencing, regardless of loop sequence andlength.

Exemplary NtHMA RNAi constructs for down-regulating the expression levelof the NtHMA gene (SEQ ID NO:1) and other NtHMA-related genes includethe following:

Various embodiments are directed to NtHMA RNAi expression vectorscomprising a NtHMA RNAi construct that comprises one or more of: intron1 (SEQ ID NO:4), exon 1 (SEQ ID NO:5), intron 2 (SEQ ID NO:6), exon 2(SEQ ID NO:7), intron 3 (SEQ ID NO:8), exon 3 (SEQ ID NO:9), intron 4(SEQ ID NO:10), exon 4 (SEQ ID NO:11), intron 5 (SEQ ID NO:12), exon 5(SEQ ID NO:13), intron 6 (SEQ ID NO:14), exon 6 (SEQ ID NO:15), intron 7(SEQ ID NO:16), exon 7 (SEQ ID NO:17), intron 8 (SEQ ID NO:18), exon 8(SEQ ID NO:19), intron 9 (SEQ ID NO:20), exon 9 (SEQ ID NO:21), intron10 (SEQ ID NO:22), exon 10 (SEQ ID NO:23), intron 11 (SEQ ID NO:24, exon11 (SEQ ID NO:25), and intron 12 (SEQ ID NO:26), fragments thereof, andvariants thereof.

Various embodiments are directed to NtHMA RNAi expression vectorscomprising: a NtHMA RNAi construct having at least 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, and 99% sequence identity to a sequenceselected from the group consisting of: exon 1 (SEQ ID NO:5), a fragmentof exon 1 (SEQ ID NO:5), exon 2 (SEQ ID NO:7), a fragment of exon 2 (SEQID NO:7), exon 3 (SEQ ID NO:9), a fragment of exon 3 (SEQ ID NO:9), exon4 (SEQ ID NO:11), a fragment of exon 4 (SEQ ID NO:11), exon 5 (SEQ IDNO:13), a fragment of exon 5 (SEQ ID NO:13), exon 6 (SEQ ID NO:15), afragment of exon 6 (SEQ ID NO:15), exon 7 (SEQ ID NO:17), a fragment ofexon 7 (SEQ ID NO:17), exon 8 (SEQ ID NO:19), a fragment of exon 8 (SEQID NO:19), exon 9 (SEQ ID NO:21), a fragment of exon 9 (SEQ ID NO:21),exon 10 (SEQ ID NO:23), a fragment of exon 10 (SEQ ID NO:23), exon 11(SEQ ID NO:25), and a fragment of exon 11 (SEQ ID NO:25).

Various embodiments are directed to NtHMA RNAi expression vectorscomprising: a NtHMA RNAi construct encoding NtHMA RNAi polynucleotidescapable of self-annealing to form a hairpin structure, in which the RNAiconstruct comprises (a) a first sequence having at least 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:3or SEQ ID NO:47; (b) a second sequence encoding a spacer element of theNtHMA RNAi polynucleotide that forms a loop of the hairpin structure;and (c) a third sequence comprising a reverse complementary sequence ofthe first sequence, positioned in the same orientation as the firstsequence, wherein the second sequence is positioned between the firstsequence and the third sequence, and the second sequence isoperably-linked to the first sequence and to the third sequence.

Various embodiments are directed to NtHMA RNAi expression vectorscomprising: a NtHMA RNAi construct encoding NtHMA RNAi polynucleotidescapable of self-annealing to form a hairpin structure, in which the RNAiconstruct comprises (a) a first sequence having at least 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:3or SEQ ID NO:47; (b) a second sequence encoding a spacer element of theNtHMA RNAi polynucleotide that forms a loop of the hairpin structure;and (c) a third sequence comprising a reverse complementary sequence ofthe first sequence (SEQ ID NO:46 or SEQ ID NO:48), positioned in thesame orientation as the first sequence, wherein the second sequence ispositioned between the first sequence and the third sequence, and thesecond sequence is operably-linked to the first sequence and to thethird sequence.

Various embodiments are directed to NtHMA RNAi expression vectorscomprising: a NtHMA RNAi construct that comprises a first sequencehaving “substantial similarity,” or having at least 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:3, orportions of SEQ ID NO:3. Various embodiments are directed to NtHMA RNAiexpression vectors comprising a NtHMA RNAi construct that comprises afirst sequence having “substantial similarity,” or having at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQID NO:47, or portions of SEQ ID NO:47.

Various embodiments are directed to a NtHMA RNAi expression vectorscomprising: a NtHMA RNAi construct that comprises a second sequencehaving “substantial similarity,” or having at least 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, and 99% sequence identity to a sequenceselected from the group consisting of: intron 1 (SEQ ID NO:4), afragment of intron 1 (SEQ ID NO:4), intron 2 (SEQ ID NO:6), a fragmentof intron 2 (SEQ ID NO:6), intron 3 (SEQ ID NO:8), a fragment of intron3 (SEQ ID NO:8), intron 4 (SEQ ID NO:10), a fragment of intron 4 (SEQ IDNO:10), intron 5 (SEQ ID NO:12), a fragment of intron 5 (SEQ ID NO:12),intron 6 (SEQ ID NO:14), a fragment of intron 6 (SEQ ID NO:14), intron 7(SEQ ID NO:16), a fragment of intron 7 (SEQ ID NO:16), intron 8 (SEQ IDNO:18), a fragment of intron 8 (SEQ ID NO:18), intron 9 (SEQ ID NO:20),a fragment of intron 9 (SEQ ID NO:20), intron 10 (SEQ ID NO:22), afragment of intron 10 (SEQ ID NO:22), intron 11 (SEQ ID NO:24), afragment of intron 11 (SEQ ID NO:24), intron 12 (SEQ ID NO:26), and afragment of intron 12 (SEQ ID NO:26). Alternatively, the second sequenceof the NtHMA RNAi construct can be randomly generated without utilizingan intron sequence derived from the NtHMA gene (SEQ ID NO:1).

Various embodiments are directed to NtHMA RNAi expression vectorscomprising: a NtHMA RNAi construct that comprises a third sequencehaving “substantial similarity,” or having at least 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:46, orportions of SEQ ID NO:46. Various embodiments are directed to NtHMA RNAiexpression vectors comprising a NtHMA RNAi construct that comprises athird sequence having “substantial similarity,” or having at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQID NO:48, or portions of SEQ ID NO:48.

Various embodiments are directed to NtHMA RNAi expression vectorscomprising: a NtHMA RNAi construct that comprises a third sequencehaving “substantial similarity,” or having at least 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, and 99% sequence identity to a reversecomplementary sequence selected from the group consisting of: SEQ IDNO:27 (exon 1), a fragment of SEQ ID NO:27 (exon 1), SEQ ID NO:28 (exon2), a fragment of SEQ ID NO:28 (exon 2), SEQ ID NO:29 (exon 3), afragment of SEQ ID NO:29 (exon 3), SEQ ID NO:30 (exon 4), a fragment ofSEQ ID NO:30 (exon 4), SEQ ID NO:31 (exon 5), a fragment of SEQ ID NO:31(exon 5), SEQ ID NO:32 (exon 6), a fragment of SEQ ID NO:32 (exon 6),SEQ ID NO:33 (exon 7), a fragment of SEQ ID NO:33 (exon 7), SEQ ID NO:34(exon 8), a fragment of SEQ ID NO:34 (exon 8), SEQ ID NO:35 (exon 9), afragment of SEQ ID NO:35 (exon 9), SEQ ID NO:36 (exon 10), a fragment ofSEQ ID NO:36 (exon 10), SEQ ID NO:37 (exon 11), and a fragment of SEQ IDNO:37 (exon 11).

Various embodiments are directed to NtHMA RNAi expression vectorscomprising a NtHMA RNAi construct that comprises: SEQ ID NO:38 (“sensesequence/fragment”), the second sequence comprises SEQ ID NO:39 (“spacersequence/fragment”) and the third sequence comprises SEQ ID NO:40(“anti-sense sequence/fragment”).

Various embodiments are directed to NtHMA RNAi expression vectorscomprising a NtHMA RNAi construct that comprises: SEQ ID NO:42 (“sensesequence/fragment”), the second sequence comprises SEQ ID NO:43 (“spacersequence/fragment”), and the third sequence comprises SEQ ID NO:44(“anti-sense sequence/fragment”).

Alternatively, the disclosed sequences can be utilized for constructingvarious NtHMA polynucleotides that do not form hairpin structures. Forexample, a NtHMA long double-stranded RNA can be formed by (1)transcribing a first strand of the NtHMA cDNA by operably-linking to afirst promoter, and (2) transcribing the reverse complementary sequenceof the first strand of the NtHMA cDNA fragment by operably-linking to asecond promoter. Each strand of the NtHMA polynucleotide can betranscribed from the same expression vector, or from differentexpression vectors. The NtHMA RNA duplex having RNA interferenceactivity can be enzymatically converted to siRNAs to reduce NtHMA RNAlevels.

C. Expression Vectors for Reducing NtHMA Gene Expression byCo-suppression

Various compositions and methods are provided for reducing theendogenous expression levels for members of the NtHMA gene family bypromoting co-suppression of NtHMA gene expression. The phenomenon ofco-suppression occurs as a result of introducing multiple copies of atransgene into a plant cell host. Integration of multiple copies of atransgene can result in reduced expression of the transgene and thetargeted endogenous gene. The degree of co-suppression is dependent onthe degree of sequence identity between the transgene and the targetedendogenous gene. The silencing of both the endogenous gene and thetransgene can occur by extensive methylation of the silenced loci (i.e.,the endogenous promoter and endogenous gene of interest) that canpreclude transcription. Alternatively, in some cases, co-suppression ofthe endogenous gene and the transgene can occur by post transcriptionalgene silencing (“PTGS”), in which transcripts can be produced butenhanced rates of degradation preclude accumulation of transcripts. Themechanism for co-suppression by PTGS is thought to resemble RNAinterference, in that RNA seems to be both an important initiator and atarget in these processes, and may be mediated at least in part by thesame molecular machinery, possibly through RNA-guided degradation ofmRNAs.

Co-suppression of members of the NtHMA gene family can be achieved byintegrating multiple copies of the NtHMA cDNA or fragments thereof, astransgenes, into the genome of a plant of interest. The host plant canbe transformed with an expression vector comprising a promoteroperably-linked to NtHMA cDNA or fragments thereof. Various embodimentsare directed to expression vectors for promoting co-suppression ofendogenous genes of the NtHMA family comprising: a promoteroperably-linked to NtHMA cDNA identified as Clone P6663 (SEQ ID NO:3) ora fragment thereof, or NtHMA cDNA identified as Clone P6643 (SEQ IDNO:47) or a fragment thereof. Various embodiments are directed toexpression vectors for promoting co-suppression of endogenous genes ofthe NtHMA family comprising: a promoter operably-linked to NtHMA cDNA,or a fragment thereof, having at least about 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3 or SEQ IDNO:47.

Various embodiments are directed to methods for reducing the expressionlevel of endogenous genes of the NtHMA family by integrating multiplecopies of NtHMA cDNA or a fragment thereof into a plant genome,comprising: transforming a plant cell host with an expression vectorthat comprises a promoter operably-linked to SEQ ID NO:3, or a fragmentthereof; or SEQ ID NO:47, or a fragment thereof. Various embodiments aredirected to methods for reducing the expression level of endogenousgenes of the NtHMA family by integrating multiple copies of NtHMA cDNA,or a fragment thereof, into a plant genome, comprising: transforming aplant cell host with an expression vector that comprises a promoteroperably-linked to NtHMA cDNA, or a fragment thereof, having at leastabout 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to SEQ ID NO:3 or SEQ ID NO:47.

D. Expression Vectors for Reducing NtHMA Gene Expression by Inhibitionof Translation by Anti-sense Agents

Various compositions and methods are provided for reducing theendogenous expression level of the NtHMA gene family by inhibiting thetranslation of NtHMA mRNA. A host plant cell can be transformed with anexpression vector comprising: a promoter operably-linked to NtHMA cDNAor a fragment thereof, positioned in anti-sense orientation with respectto the promoter to enable the expression of RNA polynucleotides having asequence complementary to a portion of NtHMA mRNA. Various expressionvectors for inhibiting the translation of HMA mRNA comprise: a promoteroperably-linked to NtHMA cDNA identified as Clone P6663 (SEQ ID NO:3) ora fragment thereof; or NtHMA cDNA identified as Clone P6643 (SEQ IDNO:47) or a fragment thereof, in which the NtHMA cDNA, or the fragmentthereof, is positioned in anti-sense orientation with respect to thepromoter. Various expression vectors for inhibiting the translation ofHMA mRNA comprise: a promoter operably-linked to a NtHMA cDNA, or afragment thereof, having at least about 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3 or SEQ ID NO:47,in which the NtHMA cDNA, or the fragment thereof, is positioned inanti-sense orientation with respect to the promoter. The lengths ofanti-sense NtHMA RNA polynucleotides can vary, including 15-20nucleotides, 20-30 nucleotides, 30-50 nucleotides, 50-75 nucleotides,75-100 nucleotides, 100-150 nucleotides, 150-200 nucleotides, and200-300 nucleotides.

Various embodiments are directed to methods for reducing the expressionlevel of endogenous genes of the NtHMA family by inhibiting NtHMA mRNAtranslation, comprising: transforming a plant cell host with anexpression vector that comprises a promoter operably-linked to NtHMAcDNA identified as Clone P6663 (SEQ ID NO:3) or a fragment thereof; orNtHMA cDNA identified as Clone P6643 (SEQ ID NO:47) or a fragmentthereof, in which the NtHMA cDNA, or the fragment thereof, is positionedin anti-sense orientation with respect to the promoter. Variousembodiments are directed to methods for reducing the expression level ofendogenous genes of the NtHMA family by inhibiting NtHMA mRNAtranslation, comprising: transforming a plant cell host with anexpression vector that comprises a promoter operably-linked to a NtHMAcDNA, or a fragment thereof, having at least about 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3 or SEQID NO:47, in which the NtHMA cDNA, or the fragment thereof, ispositioned in anti-sense orientation with respect to the promoter.

E. Other Compositions and Methods for Reducing NtHMA Gene Expression

Methods for obtaining conservative variants and more divergent variantsof NtHMA polynucleotides and polypeptides are known to persons skilledin the art. Any plant of interest can be genetically modified by variousmethods known to induce mutagenesis, including site-directedmutagenesis, oligonucleotide-directed mutagenesis, chemically-inducedmutagenesis, irradiation-induced mutagenesis, and other equivalentmethods. For example, site-directed mutagenesis is described in, e.g.,Smith (1985) “In vitro mutagenesis,” Ann. Rev. Genet. 19:423-462, andreferences therein, such as Botstein & Shortle (1985) “Strategies andApplications of in vitro Mutagenesis,” Science 229:1193-1201; and inCarter (1986) “Site-directed mutagenesis,” Biochem. J. 237:1-7.Oligonucleotide-directed mutagenesis is described in, e.g., Zoller &Smith (1982) “Oligonucleotide-directed Mutagenesis using M13-derivedVectors: an Efficient and General Procedure for the Production of Pointmutations in any DNA Fragment,” Nucleic Acids Res. 10:6487-6500.Mutagenesis utilizing modified bases is described in, e.g., Kunkel(1985) “Rapid and Efficient Site-specific Mutagenesis without PhenotypicSelection,” Proc. Natl. Acad. Sci. USA 82:488-492, and in Taylor et al.(1985) “The Rapid Generation of Oligonucleotide-directed Mutations atHigh Frequency using Phosphorothioate-modified DNA,” Nucl. Acids Res.13: 8765-8787. Mutagenesis utilizing gapped duplex DNA is described in,e.g., Kramer et al. (1984) “The Gapped Duplex DNA Approach toOligonucleotide-directed Mutation Construction,” Nucl. Acids Res. 12:9441-9460). Point-mismatch mutagenesis is described in, e.g., Kramer etal. (1984) “Point Mismatch Repair,” Cell 38:879-887). Double-strandbreak mutagenesis is described in, e.g., Mandecki (1986)“Oligonucleotide-directed Double-strand Break Repair in Plasmids ofEscherichia coli: A Method for Site-specific Mutagenesis,” Proc. Natl.Acad. Sci. USA, 83:7177-7181, and in Arnold (1993) “Protein Engineeringfor Unusual Environments,” Current Opinion in Biotechnology 4:450-455).Mutagenesis utilizing repair-deficient host strains is described in,e.g., Carter et al. (1985) “Improved Oligonucleotide Site-directedMutagenesis using M13 Vectors,” Nucl. Acids Res. 13: 4431-4443.Mutagenesis by total gene synthesis is described in, e.g., Nambiar etal. (1984) “Total Synthesis and Cloning of a Gene Coding for theRibonuclease S Protein,” Science 223: 1299-1301. DNA shuffling isdescribed in, e.g., Stemmer (1994) “Rapid Evolution of a Protein invitro by DNA Shuffling,” Nature 370:389-391, and in Stemmer (1994) “DNAshuffling by random fragmentation and reassembly: In Vitro Recombinationfor Molecular Evolution,” Proc. Natl. Acad. Sci. USA 91:10747-10751.

Alternatively, NtHMA genes can be targeted for inactivation byintroducing transposons (and IS elements) into the genomes of plants ofinterest. These mobile genetic elements can be introduced by sexualcross-fertilization and insertion mutants can be screened for loss inNtHMA activity, such as reduced Cd transport. The disrupted NtHMA genein a parent plant can be introduced into other plants by crossing theparent plant with plant not subjected to transposon-induced mutagenesisby, e.g., sexual cross-fertilization. Any standard breeding techniquesknown to persons skilled in the art can be utilized. In one embodiment,one or more NtHMA-related genes can be inactivated by the insertion ofone or more transposons. Mutations can result in homozygous disruptionof one or more NtHMA genes, in heterozygous disruption of one or moreNtHMA genes, or a combination of both homozygous and heterozygousdisruptions if more than one NtHMA gene is disrupted. Suitabletransposable elements can be selected from two broad classes, designatedas Class I and Class II. Suitable Class I transposable elements includeretrotransposons, retroposons, and SINE-like elements. Such methods areknown to persons skilled in the art as described in Kumar and Bennetzen(1999), Plant Retrotransposons in Annual Review of Genetics 33:479.

Alternatively, NtHMA genes can be targeted for inactivation by a methodreferred to as Targeting Induced Local Lesions IN Genomics (“TILLING”),which combines high-density point mutations with rapid sensitivedetection of mutations. Typically, plant seeds are exposed to mutagens,such as ethylmethanesulfonate (EMS) or EMS alkylates guanine, whichtypically leads to mispairing. Suitable agents and methods are known topersons skilled in the art as described in McCallum et al., (2000),“Targeting Induced Local Lesions IN Genomics (TILLING) for PlantFunctional Genomics,” Plant Physiology 123:439-442; McCallum et al.,(2000) “Targeted screening for induced mutations,” Nature Biotechnology18:455-457; and Colbert et al., (2001) “High-Throughput Screening forInduced Point Mutations,” Plant Physiology 126:480-484.

Alternatively, NtHMA genes can be targeted for inactivation byintroducing ribozymes derived from a number of small circular RNAs thatare capable of self-cleavage and replication in plants. These RNAs canreplicate either alone (viroid RNAs) or with a helper virus (satelliteRNAs). Examples of suitable RNAs include those derived from avocadosunblotch viroid and satellite RNAs derived from tobacco ringspot virus,lucerne transient streak virus, velvet tobacco mottle virus, solanumnodiflorum mottle virus, and subterranean clover mottle virus. Varioustarget RNA-specific ribozymes are known to persons skilled in the art asdescribed in Haseloff et al. (1988) Nature, 334:585-591.

III. Transgenic Plants, Cell Lines, and Seeds Comprising NtHMA RNAiPolynucleotides and Related Methods

Various embodiments are directed to transgenic plants geneticallymodified to reduce the NtHMA gene expression level by various methodsthat can utilized for silencing NtHMA gene expression, and thereby,producing transgenic plants in which the expression level of NtHMAtransporters can be reduced within plant tissues of interest. Rates ofheavy metal transport and distribution patterns of heavy metaltransport, in particular, cadmium transport, can be altered intransgenic plants produced according to the disclosed methods andcompositions. Plants suitable for genetic modification include monocotsand dicots.

Various embodiments are directed to transgenic tobacco plantsgenetically modified to reduce the NtHMA gene expression level byvarious methods, known to persons skilled in the art, that can beutilized for down-regulating NtHMA gene expression, and thereby,producing transgenic tobacco plants in which the expression level ofNtHMA transporters can be reduced within plant tissues of interest.Various expression vectors have been provided to produce transgeniclines of tobacco of any variety exhibiting reduced levels of NtHMA geneexpression. The disclosed compositions and methods can be applied to anyplant species of interest, including plants of the genus Nicotiana,various species of Nicotiana, including N. rustica and N. tabacum (e.g.,LA B21, LN KY171, TI 1406, Basma, Galpao, Perique, Beinhart 1000-1, andPetico). Other species include N. acaulis, N. acuminata, N. acuminatavar. multiflora, N. africana, N. alata, N. amplexicaulis, N. arentsii,N. attenuata, N. benavidesii, N. benthamiana, N. bigelovii, N.bonariensis, N. cavicola, N. clevelandii, N. cordifolia, N. corymbosa,N. debneyi, N. excelsior, N. forgetiana, N. fragrans, N. glauca, N.glutinosa, N. goodspeedii, N. gossei, N. hybrid, N. ingulba, N.kawakamii, N. knightiana, N. langsdorffii, N. linearis, N. longiflora,N. maritima, N. megalosiphon, N. miersii, N. noctiflora, N. nudicaulis,N. obtusifolia, N. occidentalis, N. occidentalis subsp. hesperis, N.otophora, N. paniculata, N. pauciflora, N. petunioides, N.plumbaginifolia, N. quadrivalvis, N. raimondii, N. repanda, N. rosulata,N. rosulata subsp. ingulba, N. rotundifolia, N. setchellii, N. simulans,N. solanifolia, N. spegazzinii, N. stocktonii, N. suaveolens, N.sylvestris, N. thyrsiflora, N. tomentosa, N. tomentosiformis, N.trigonophylla, N. umbratica, N. undulata, N. velutina, N. wigandioides,and N. x sanderae. Suitable plants for transformation include any planttissue capable of transformation by various methods of transformingplants known by persons skilled in the art, including electroporation,micro-projectile bombardment, and Agrobacterium-mediated transfer asdescribed, for example, in U.S. Pat. No. 4,459,355 that discloses amethod for transforming susceptible plants, including dicots, with anAgrobacterium strain containing a Ti plasmid; U.S. Pat. No. 4,795,855that discloses transformation of woody plants with an Agrobacteriumvector; U.S. Pat. No. 4,940,838 that discloses a binary Agrobacteriumvector; U.S. Pat. No. 4,945,050; and U.S. Pat. No. 5,015,580.

Various embodiments are directed to transgenic tobacco plantsgenetically modified to exogenously express a RNAi construct encodingNtHMA RNAi polynucleotides that facilitate the degradation of NtHMA RNAtranscripts, and consequently, that reduce the number of RNA transcriptsavailable for translation into NtHMA transporters. Various embodimentsare directed to transgenic plants comprising an expression vector thatenable the expression of NtHMA polynucleotides produced according to thedisclosed methods. Various embodiments are directed to cell linesderived from transgenic plants produced according to the disclosedmethods. Various embodiments are directed to transgenic seeds derivedfrom transgenic plants produced according to the disclosed methods.

Various embodiments are directed to methods for reducing the NtHMA geneexpression levels in plants, the method comprising reducing theexpression level of a NtHMA gene, which can be accomplished by variousmethods known to persons skilled in the art. As examples, thisdisclosure described: (1) RNA interference method for reducingsteady-state level of endogenous NtHMA RNA variants available fortranslation by expression of NtHMA RNAi polynucleotides; (2)co-suppression method for reducing transcription of NtHMA gene(s) byintegrating multiple copies of the NtHMA cDNA or fragments thereof, astransgenes, into a plant genome; (3) anti-sense method for reducing theNtHMA translation by the expression of anti-sense polynucleotides thatcan target NtHMA RNA; and (4) various methods for inducing mutagenesis.

Various embodiments are directed to transgenic tobacco plantsgenetically modified to reduce the NtHMA gene expression level byvarious methods, known to persons skilled in the art, and furthermodified either to reduce the expression of a second endogenous gene ofinterest (i.e., not NtHMA-related) or to enhance the expression of anexogenous gene of interest (i.e., not NtHMA-related). For example, thedown-regulation of a second endogenous gene of interest encoding anenzyme involved in the biosynthesis of alkyloids may be desirable. Inother situations, the enhancement in the expression level of a transgeneencoding a recombinant protein of interest, such as a human hormone fortherapeutic use, may be desirable. Persons skilled in the art arecapable of producing various transgenic plants that can be modified, forexample, to exogenously express NtHMA RNAi polynucleotides and at leastone recombinant gene product of interest, such as a recombinant humangrowth factor or RNAi polynucleotides that can target a second gene ofinterest not related to the NtHMA family.

Producing transgenic plants according to the disclosed methods providesa number of advantages. Transgenic plants, including transgenic tobaccoplants, can be grown in soils containing variable Cd concentrations, orin soils containing less than desirable Cd concentrations. Thesetransgenic plants and derivative seeds can provide more options forcultivating them in a broader range of soil environments, which mayincrease the amount of cultivatable soils available to practitioners(e.g., farmers). Furthermore, these transgenic plants, exhibitingreduced Cd content, compared to non-transgenic counterparts can beconsumed directly as edible products. The consumption of edible portionsof these transgenic plants can be a healthier option compared to theconsumption of non-transgenic counterparts. Suitable plants that can begenetically modified according to the disclosed methods, include plantscultivatable for agricultural use, including rice, corn, squash,soybeans, lettuce, potatoes, beats, herbs, wheat, barley, carrots, etc.The % Cd reduction in these transgenic plants, including the leaf laminaportion, can be approximately at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, and 99%, when compared to non-transgenic counterparts. The Cdcontent of these transgenic plants, including the leaf lamina portion,is a value from a range from about 0.01 to about 0.05 ppm, from about0.01 to about 0.1 ppm, from about 0.01 to about 0.5 ppm, from about 0.01to about 1.0 ppm, and from about 0.01 to about 5 ppm.

IV. Consumable Products Incorporating Tobacco Leaves GeneticallyModified to Contain Reduced Cd Content

Various embodiments provide transgenic plants, in which the expressionlevel of members of the NtHMA gene family is substantially reduced tocurtail or impede Cd transport into the leaf lamina. The leaf laminaderived from transgenic tobacco plants, produced according to thedisclosed methods, can be incorporated into various consumable productscontaining Cd at a level substantially below that of consumable productsmade by incorporating tobacco leaves derived from plants of the samegenotype that were grown under identical conditions, but not geneticallymodified with respect to the reduced expression level of members of theNtHMA gene family (“non-transgenic counterparts”).

In some embodiments, these transgenic plants exhibiting reduced Cdcontent compared to non-transgenic counterparts can be incorporated intoconsumable products, including various smokable articles, such ascigars, cigarettes, and smokeless tobacco products (i.e.,non-combustible). Smokable articles and smokeless tobacco products,produced by incorporating tobacco leaves derived from tobacco plantsgenetically modified to contain reduced Cd levels according to thedisclosed methods, can provide healthier options compared tonon-transgenic counterparts.

Smokeless tobacco products incorporating tobacco plants geneticallymodified according to the disclosed methods can be manufactured in anyformat suitable for comfort in a consumer's oral cavity. Smokelesstobacco products contain tobacco in any form, including as driedparticles, shreds, granules, powders, or a slurry (i.e., tobaccoextract), deposited on, mixed in, surrounded by, or otherwise combinedwith other ingredients in any format, such as flakes, films, tabs,foams, or beads. Smokeless tobacco products may be wrapped with amaterial, which may be edible (i.e., orally disintegrable) or nonedible.Liquid contents of smokeless tobacco products can be enclosed in a form,such as beads, to preclude interaction with a water-soluble wrapper. Thewrapper may be shaped as a pouch to partially or completely enclosetobacco-incorporating compositions, or to function as an adhesive tohold together a plurality of tabs, beads, or flakes of tobacco. Awrapper may also enclose a moldable tobacco composition that conforms tothe shape of a consumer's mouth. An orally disintegrable wrapper mayenclose smokeless tobacco, e.g., as dry snuff or soluble tobacco, andmay be formed on continuous thermoforming or horizontal form/fill/sealequipment or other suitable packaging equipment using edible films(which may or may not contain tobacco). Exemplary materials forconstructing a wrapper include film compositions comprising HPMC, CMC,pectin, alginates, pullulan, and other commercially viable, ediblefilm-forming polymers. Other wrapping materials may include pre-formedcapsules produced from gelatin, HPMC, starch/carrageenan, or othercommercially available materials. Such wrapping materials may includetobacco as an ingredient. Wrappers that are not orally disintegrable maybe composed of woven or nonwoven fabrics, of coated or uncoated paper,or of perforated or otherwise porous plastic films. Wrappers mayincorporate flavoring and/or coloring agents. Smokeless products can beassembled together with a wrapper utilizing any method known to personsskilled in the art of commercial packaging, including methods such asblister packing and stik-paking, in which a small package can be formedby a vertical form/fill/seal packaging machine.

The % Cd reduction in these smokable articles and smokeless products,produced by incorporating tobacco leaves derived from tobacco plantsgenetically modified to contain reduced Cd levels, is a value of atleast about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, and 100%, whencompared to consumable products derived from non-transgeniccounterparts. In some embodiments, the Cd content of these smokablearticles and smokeless products, produced by incorporating tobaccoleaves derived from tobacco plants genetically modified to containreduced Cd levels, is a value from a range from about 0.01 to about 0.05ppm, from about 0.01 to about 0.1 ppm, from about 0.01 to about 0.5 ppm,from about 0.01 to about 1.0 ppm, and from about 0.01 to about 5 ppm.

The degree of Cd accumulation in plants can be substantially variabledepending on several parameters attributed to the complexity of thegenotype and the growth environment. For example, Cd concentrations infield-grown tobacco leaves can be extremely variable depending onfactors such as the agro-climate, soil parameters, and cultivars.Furthermore, the relative Cd distribution patterns within differentportions of a tobacco plant can vary according to the species, theorgan/tissue, and growth conditions (i.e., field-grown vs.hydroponically-grown). On average, the Cd concentrations measured infield-grown tobacco leaves (including midribs and veins) can be in therange from approximately 0.5 to 5 ppm (parts per million, or ug/g of dryweight of tobacco leaves). However, many published Cd levels typicallydo not define the tobacco maturity stage, the tobacco variety, or theparticular leaf portions (i.e., removal from leaf stalk position)harvested for analysis. In some varieties, the lower leaves mayaccumulate higher Cd levels than the medium and upper leaves. At theintracellular level, Cd can be found in various cell components of aplant cell, including the cell wall, cytoplasm, chloroplast, nucleus,and vacuoles.

Furthermore, Cd content measured in tobacco leaves can varysubstantially depending on the Cd levels in the soil environment wherethe tobacco plants were grown. The leaves of tobacco grown inCd-contaminated areas can accumulate Cd from about 35 ppm or higher,compared to the leaves of genetically identical counterparts grown innon-contaminated areas, which can accumulate Cd at a range fromapproximately 0.4 to approximately 8 ppm. The vacuoles within the leavesof plants grown in Cd-contaminated areas can accumulate very high Cdconcentrations. Methods for modifying the disclosed compositions to besuitable for a given plant species of interest are known to personsskilled in the art.

EXAMPLES Example 1 Cloning and Exon Mapping of a Full-Length NicotianaNtHMA Genomic Clone

Two partial genomic clones representing different portions of anendogenous NtHMA gene were independently identified, referred to as“CHO_OF96xf01.ab1” and “CHO_OF261xo09c1.ab1.” Based on sequenceinformation obtained from the partial genomic clones, a full-lengthgenomic clone (_HO-18-2) and 4 full-length NtHMA cDNAs were subsequentlyidentified, including clone P6663 (SEQ ID NO:3) and clone P6643 (SEQ IDNO:47). The exon and intron subregions of full-length genomic clone(_HO-18-2) (17,921 bp) were mapped. As shown in FIG. 1A, thefull-length, endogenous NtHMA gene cloned from Nicotiana comprises 11exons consisting of 3392 nucleotides in total.

Example 2 Construction of NtHMA RNAi Expression Vector PBI121-NtHMA(660-915) Encoding RNAi Polynucleotides

FIG. 1B provides a list of nucleotide positions mapped to each exonwithin the isolated NtHMA genomic clone (SEQ ID NO: 1) (“Table 1”). Thepartial genomic clone CHO_OF96xf01.ab1 includes a part of intron 4, exon4, intron 5, exon 5, intron 6, and a part of exon 6, as shown in FIG.1A, and listed under Table 1 of FIG. 1B. The partial genomic cloneCHO_OF261 xo09c1 includes a part of intron 7, exon 7, intron 8, exon 8,and a part of exon 9, as shown in FIG. 1A. To produce transgenic plantsthat can stably produce recombinant NtHMA RNAi polynucleotides ofinterest that can facilitate the degradation of endogenous RNAtranscripts encoding NtHMA polypeptides, two sets of NtHMA RNAiexpression vectors, the PBI121-NtHMA (660-915) RNAi expression vector asfurther described below, and the PBI121-NtHMA (1382-1584) RNAiexpression vector as further described in Example 3.

FIG. 2 illustrates an exemplary subcloning strategy for constructing aNtHMA RNAi expression vector that enables the constitutive expression ofNtHMA RNAi polynucleotides of interest. Based on exon mapping andsequence analysis of genomic clone CHO_OF96xf01.ab1, RNAi constructswere designed.

FIG. 3A shows an exemplary RNAi sequence, NtHMA (660-915), for producingNtHMA RNAi polynucleotides of interest. In FIG. 3A, NtHMA RNAi RNAiconstruct comprises a sense fragment (272 bp) (SEQ ID NO:38) composed ofexon 4 (272 bp), which is positioned upstream and operably-linked to aspacer fragment (80 bp) (SEQ ID NO:39) composed of intron 5, which ispositioned upstream and operably-linked to a reverse complementaryfragment (272 bp) (SEQ ID NO:40) composed of exon 4 positioned inanti-sense orientation. RNAi constructs encoding NtHMA RNAipolynucleotides of interest were inserted into the PBKCMV cloningvector, and were placed downstream and operably-linked to acytomegalovirus (CMV) promoter. XbaI and HindIII sites were incorporatedinto the 5′ and 3′ ends of the 352 bp NtHMA sense fragment, whichincluded the 80 bp intron fragment by utilizing PCR primers modified toincorporate these restriction enzyme sites (PMG783F:ATTCTAGACTGCTGCTATGTCATCACTGG (SEQ ID NO:54) and PMG783R:ATAAGCTTAGCCTGAAGAATTGAGCAAA (SEQ ID NO:55)). Similarly, SpeI and SacIsites were incorporated into the 5′ and 3′ ends of the correspondingNtHMA reverse complementary fragment by utilizing PCR primers (PMG 785F:ATGAGCTCTGGTTATGTAGGCTACTGCTGCT (SEQ ID NO:56) and PMG 786R:ATACTAGTATTTGTAGTGCCAGCCCAGA (SEQ ID NO:57)) to produce the PBKCMV-NtHMARNAi plasmid. The PBI121-NtHMA RNAi expression vectors were constructedby (a) excising the β-glucuronidase ORF from the binary expressionvector (“pBI121” from CLONTECH), and (b) substituting the NtHMA RNAiconstruct, excised from the PBKCMV-NtHMA RNAi plasmid, into XbaI/SacIsites of the PBI121 plasmid in place of the removed β-glucuronidase ORF.The PBI121-NtHMA RNAi expression vectors comprise: (i) 352 bpXbaI-HindIII NtHMA sense fragment that includes (ii) 80 bp intronfragment, operably-linked to the (iii) 272 bp SpeII-SacI NtHMA reversecomplementary fragment.

Example 3 Construction of NtHMA RNAi Expression Vector PBI121-NtHMA(1382-1584) Encoding RNAi Polynucleotides

FIG. 4A shows an exemplary RNAi sequence, NtHMA (1382-1584), forproducing NtHMA RNAi polynucleotides of interest. Based on exon mappingand sequence analysis of genomic clone CHO_OF261xo09c1, a RNAi constructwas designed that includes a sense fragment (191 bp) (SEQ ID NO:42)comprising sequences of exon 7, which is positioned upstream andoperably-linked to a spacer DNA fragment (139 bp) (SEQ ID NO:43)comprising sequences of intron 8, which is positioned upstream andoperably-linked to a reverse complementary fragment (196 bp) (SEQ IDNO:44) comprising sequences of exon 7 positioned in anti-senseorientation. These RNAi constructs encoding NtHMA RNAi polynucleotidesof interest were inserted into the PBKCMV cloning vector, and wereplaced downstream and operably-linked to a cytomegalovirus (CMV)promoter. XbaI and HindIII sites were incorporated into the 5′ and 3′ends of the 330 bp NtHMA sense fragment, which included the 139 bpintron fragment by utilizing PCR primers modified to incorporate theserestriction enzyme sites (PMG754F: ATTCTAGATGAGAGCAAGTCAGGTCATCC (SEQ IDNO:58) and PMG754R: ATAAGCTTTTCAAACATCCACCGCATTA (SEQ ID NO:59)).Similarly, PstI and SacI sites were incorporated into the 5′ and 3′ endsof the corresponding NtHMA reverse complementary fragment by utilizingPCR primers PMG757F: ATGAGCTCGCATTGAGAGCAAGTCAGGTC (SEQ ID NO:60) andPMG757R: ATCTGCAGCCTGTGGTACATCCAGCTCTT (SEQ ID NO:61)) to produce thePBKCMV-NtHMA RNAi expression vector.

The PBI121-NtHMA RNAi expression vectors were constructed by (a)excising the β-glucuronidase ORF from the binary expression vector(“pBI121” from CLONTECH), and (b) substituting the NtHMA RNAi construct,excised from the PBKCMV-NtHMA RNAi plasmid, into XbaI/SacI sites of thePBI121 plasmid in place of the removed β-glucuronidase ORF. ThePBI121-NtHMA RNAi expression vectors comprise: (i) 330 bp XbaI-HindIIINtHMA sense fragment that includes (ii) 139 bp intron fragment,operably-linked to the (iii) 196 bp SpeII-SacI NtHMA reversecomplementary fragment. The PBI121-NtHMA RNAi expression vectors, suchas those described in Examples 2 and 3, can be introduced into any hostplant cell of interest by various methods known to persons skilled inthe art.

Example 4 Transformation of Burley (TN90), Flue-Cured (K326), and Dark(VA359) Tobacco Varieties with NtHMA RNAi Expression Vectors

Tobacco seeds from three different varieties, Burley (TN90), Flue-cured(K326), and Dark (VA359), were sterilized and germinated in a petridishcontaining MS basal media supplemented with 5 ml/L plant preservativemixture (PPM). Seedlings, at approximately 7 to 10 dayspost-germination, were selected for transformation with various NtHMARNAi expression vectors. A single colony of Agrobacterium tumefaciensLBA4404 was inoculated into a liquid LB medium containing 50 mg l⁻¹kanamycin (kanamycin mono sulphate), and were incubated for 48 h at 28°C. with reciprocal shaking (150 cycles min⁻¹). Cultured bacterial cellswere collected by centrifugation (6000×g, 10 min), and were suspended toa final density of 0.4-0.7 OD₆₀₀, with 20 ml liquid MS medium containing20 g⁻¹ sucrose. The 7-10 day seedling explants were immersed into abacterial suspension for 5 mins, and were blotted on sterile filterpapers. Fifty explants were placed onto 40 ml aliquots of REG agarmedium (MS basal medium supplemented with 0.1 mg l⁻¹ NAA and 1 mg l⁻¹BAP) in 100 mm×20 mm petri dishes. The explants were co-cultivated withAgrobacterium at 25° C. After 3 days of co-cultivation, the explantswere washed and transferred to RCPK medium (REG medium with 100 mg⁻¹kanamycin, 500 mg l⁻¹ carbenicillin, and 5 ml PPM) to select fortransformants. The explants were subcultured every 2 weeks. After 8-12weeks of growth under selective conditions, the surviving plants,representing transformants that have integrated the NtHMA RNAiexpression constructs into their genomes, were transferred to a rootingmedium (MS basal medium supplemented with 100 mg l⁻¹ Kanamycin). Rootedplants were transferred to pots to promote further growth.

Example 5 Cd Reduction in Leaf Lamina of First Generation TransgenicsGenetically Modified to Express NtHMA RNAi Polynucleotides

To determine the effect of NtHMA RNAi polynucleotide expression on Cdtransport from the root to aerial portions of transgenic plants, the Cdlevels were determined for several transgenic lines that have beengenetically modified to express either the NtHMA (660-915) or the(1382-1584) RNAi polynucleotides.

Approximately 40 independent transgenic plants, representing threetobacco varieties, were transformed with various PBI121-NtHMA RNAiexpression vectors. Initially, transformants were grown in floatingtrays containing Hoaglands medium for 4 weeks. PCR positive plants forNPT II were selected and potted in 10″ pots with a hydroponic systemcontaining Hoaglands medium containing 5 μM CdCl₂. After 4-8 weeks, twomiddle leaves samples were harvested and freeze-dried for metalanalysis, or were frozen in liquid nitrogen for gene expressionanalysis. Approximately 500 mg of tobacco was weighed and digested in 10ml of concentrated HNO₃ by microwave-accelerated, reaction system 5digestion system (CEM corporation, Mathews, N.C.). Heavy metalconcentrations were analyzed utilizing inductively coupled plasma-massspectrophotometry (“ICP-MS,” Agilent 7500A; Agilent Technologies, PaloAlto, Calif.). As non-transgenic tobacco control, a sample consisting ofpolish-certified, Virginia tobacco leaves, CTA-VTL-2, was prepared undercomparable conditions (Dybczynski et al., 1997).

FIGS. 3B-3D show Cd reduction in leaf lamina of multiple firstgeneration (T0) transgenic lines, representing three varieties, thathave been genetically modified to express NtHMA RNAi polynucleotides(660-915).

FIGS. 4B-4D show Cd reduction in leaf lamina of multiple firstgeneration (T0) transgenic lines, representing three varieties, thathave been genetically modified to express NtHMA RNAi polynucleotides(1382-1584).

Example 6 Reduction in NtHMA RNA Transcripts in Transgenic Tobacco Leafby the Expression of NtHMA RNAi Polynucleotides

To determine the effect of NtHMA RNAi polynucleotide expression on thesteady-state levels of endogenous NtHMA RNA transcripts, the relativechange in NtHMA RNA transcripts was measured by isolating total cellularRNA from leaf lamina portions of various transgenic lines, representingthree tobacco varieties.

Total RNA was isolated from middle leaves of T0 plants using TRIOReagent (Sigma-Aldrich, St. Louis, Mo.). To remove DNA impurities,purified RNA was treated with RNase-free DNase (TURBO DNA-free, Ambion,Austin Tex.). To synthesize the first cDNA strand, approximately 10 μgof total RNA was reverse transcribed utilizing the High Capacity cDNAArchive Kit (Applied Biosystems, Foster City, Calif.). To measure thelevel of NtHMA transcripts in the samples, a quantitative 2-step RT-PCRwas performed according to the Taqman MGB probe-based chemistry. The RTmixture contained 4 μM dNTP mix, 1× random primers, 1×RT Buffer, 10 gcDNA, 50 U Multiscribe Reverse transcriptase (Applied Biosystems), 2 USuperase-In RNase Inhibitor (Ambion), and nuclease-free water. The PCRmixture contained 1× Taqman Universal PCR Master Mix (AppliedBiosystems, Foster City, Calif.), 400 nM forward primer, 400 nM reverseprimer, 250 nM Taqman MGB probe, 2 ng of cDNA, and nuclease-free water.RT-PCR was performed utilizing an ABI 7500 Real-Time System (AppliedBiosystems, Foster City, Calif.) and under amplification conditions: 50°C. for 2 min.; 95° C. for 10 min.; 40 cycles of 95° C. for 15 sec.; and60° C. for 1 min. For normalizing the measured NtHMA RNA transcriptlevels, the Glyceraldehyde-3-Phosphate Dehydrogenase (G3PDH) wasselected as a control endogenous RNA transcript, whose expression levelis not responsive to the sequence-specific RNA interference activity ofthe NtHMA RNAi polynucleotides under analysis. The fold change in NtHMARNA transcript level caused by NtHMA-RNAi-polynucleotide expression wascalculated by determining the ratio of (a)/(b), in which (a) representsthe normalized value of NtHMA RNA transcript level determined forsamples derived from transgenic plants transformed with a NtHMA RNAiexpression vector, and (b) represents the normalized value of NtHMA RNAtranscript level determined for samples derived from transgenic plantstransformed with a control expression vector deficient in the NtHMA RNAiRNAi construct.

FIGS. 5A-C show normalized NtHMA RNA transcript levels in various firstgeneration (T0) transgenic lines that have been genetically modified toexpress NtHMA RNAi polynucleotides of interest, as determined byquantitative realtime PCR analysis of leaf lamina extracts. FIG. 5Ashows that for multiple independently derived K326 transgenic lines, theRNA transcript levels were reduced by the RNA interference activity ofNtHMA (660-915) RNAi polynucleotides. FIG. 5B shows that for multiple,independently derived TN90 transgenic lines, the RNA transcript levelswere reduced by the RNA interference activity of NtHMA (660-915) RNAipolynucleotides. FIG. 5C shows that for multiple independently derivedVA359 transgenic lines, the RNA transcript levels were reduced by theRNA interference activity of NtHMA (660-915) RNAi polynucleotides. Thereduction in NtHMA RNA transcript levels is consistent with thereduction in Cd content measured in the middle leaves for the sametransgenic lines tested. “PBI121” represents an expression vectordeficient in the RNAi construct encoding NtHMA (660-915) RNAipolynucleotides.

Example 7 Distribution of Cd and Zn in Transgenic Lines GeneticallyModified to Express NtHMA RNAi Polynucleotides

To determine the effect of NtHMA (660-915) RNAi polynucleotideexpression on the distribution of Cd and Zn within the leaf lamina andthe root, the metal content of transgenic plants of three varieties wereanalyzed. Five transgenic lines of each variety, i.e., Flue-cured(K326), Burley (TN90), and Dark (VA359), were selected for exhibiting Cdcontent at the lowest range in the leaf lamina. The middle leaves androots of these transgenic plants and control plants were harvested formetal analysis by ICP_MS. For 8 weeks, all plants were grown inHoaglands medium supplemented with 5 μM CdCl₂ prior to harvesting.

Table 2 lists Cd and Zn levels measured in the leaf lamina and the rootof several transgenic lines, representing three tobacco varieties, asprovided below. In Table 2, the Cd distribution between the leaf laminaand the root were substantially modified by the expression of NtHMA(660-915) RNAi polynucleotides for all three varieties, Flue-cured(K326), Burley (TN90), and Dark (VA359). For the K326 transgenic lines,the % Cd reduction ranged from 97.16-98.54% when compared to Cd levelsobserved in K326 Control plants. For TN90 transgenic lines, the % Cdreduction ranged from 85.12-90.96% when compared to Cd levels observedin the TN90 Control. For VA359 transgenic lines, the % Cd reductionranged from 93.24-99.07% when compared to Cd levels observed in theVA359 Control. The VA359 NtHMA-11 transgenic line exhibited the lowestCd level (1.62 μg/g) and the highest % Cd reduction (99.07%), whencompared against two NtHMA RNAi transgene-deficient control lines(“VA359 PBI121”) that exhibited Cd levels at 158.3-205.96 μg/g.Comparable root analysis of the transgenic lines showed, that asubstantial amount of Cd can accumulate in the root, resulting in foldincrease in root Cd levels ranging from 6.90-15.38, relative to the Cdlevels observed in the respective controls.

In contrast to the significant Cd reduction in the leaf lamina oftransgenic lines, the Zn content of the leaf lamina was notsubstantially reduced, although some reduction was observed in mosttransgenic lines, caused by the expression of NtHMA (660-915) RNAipolynucleotides. The Zn content within the root (last column of Table 3)increased in all transgenic lines, resulting in a 4-6 fold increase inthe transgenic lines of the K326 and VA359 varieties, and a 3-5 foldincrease in the TN90 variety.

FIG. 6 shows the distribution of Cd and Zn between the leaf lamina andthe root of various first generation transgenic lines that have beengenetically modified to express NtHMA RNAi polynucleotides of interest,as presented in Table 2.

TABLE 2 Leaf Root Transgenic Cd Zn Cd Zn Variety μg/g μg/g μg/g μg/gK326 06T458 7.09 22.2 703 201 K326 06T459 4.97 24.1 696 225 K326 06T4733.7 34 929 215 K326 06T480 3.93 38.6 989 224 K326 06T482 2.55 36.3 520126 K326 Control 174.7 36.3 64.3 35.7 TN90 06T428 26.3 48.6 626 184 TN9006T430 16.08 37.2 684 213 TN90 06T444 15.98 28.1 738 234 TN90 06T44520.72 32.6 618 186 TN90 06T455 17.87 24.4 582 157 TN90 PBI121 181.2 35.562.6 44.3 TN90 Control 172.4 32.3 72.9 46.6 VA359 06T493 7.59 23.1 543148 VA359 06T498 1.62 26.2 706 175 VA359 06T506 5.72 28.8 351 109 VA35906T542 7.03 27.1 738 136 VA359 06T543 11.78 29.3 547 106 VA359 PBI121206 47.5 35.3 27.6 VA359 Control 158.5 32.6 37.6 26.2

Example 8 Cd Distribution in Various Tissues of Transgenic LinesGenetically Modified to Express NtHMA RNAi Polynucleotides

To determine the effect of NtHMA (1382-1584) RNAi polynucleotideexpression on Cd distribution within various tissues (i.e., the bark,lamina, pith, and root), the metal content of several transgenic linesrepresenting two varieties, Burley (TN90) and Flue-cured (K326), wereanalyzed. Fully matured transgenic plants and control plants wereharvested for metal analysis by ICP_MS. For 8 weeks, all plants weregrown in 5 μM CdCl₂ in Hoaglands medium prior to harvesting.

Table 3 lists Cd content in the bark, lamina, pith, and root tissues ofseveral transgenic lines, as provided below. In Table 3, Cd levels weresubstantially reduced in the bark, lamina, and pith tissues of alltransgenic lines tested when compared that of control plants. The“Control” represents non-transgenic plants. The “PBI121” representstransgenic plants transformed with an expression vector deficient inNtHMA RNAi RNAi construct. The extent of Cd reduction in the bark, pith,and leaf lamina of K326 transgenic lines was significantly greater thanthat observed in TN90 transgenic lines. The expression of RNAi(1382-1584) polynucleotides in K326 transgenic plants resulted in a 9-11fold Cd reduction in the bark, a 6-13 fold Cd reduction in the pith, anda 31-32 fold Cd reduction in the leaf lamina. The expression of RNAi(1382-1584) polynucleotides in TN90 transgenic plants resulted in a 4-7fold Cd reduction in the bark, a 5-8 fold Cd reduction in the pith, anda 6-20 fold Cd reduction in the leaf lamina. In contrast, more modestincreases (5-6 fold) in Cd content in the root of these transgenic lineswere observed when compared to that of control plants.

FIG. 7 shows Cd distribution among the bark, leaf lamina, pith, and theroot of various first generation transgenic lines that have beengenetically modified to express NtHMA RNAi polynucleotides of interest,as presented in Table 3.

TABLE 3 Transgenic Seed Variety Bark Cd Lamina Cd Pith Cd Root Cd TN9006T619 7.36 31.1 4.67 557 TN90 06T658 3.76 8.89 2.89 727 TN90 Control30.9 151 25.3 115 TN90 PBI121 23.1 201 20 124 K326 06T682 2.02 4.32 1.971020 K326 06T696 2.53 4.48 4.25 1030 K326 Control 19.5 133 25.3 145 K326PBI121 25.5 143 26.2 253

Example 9 Cd Reduction in Leaf Lamina of Second Generation TransgenicLines Genetically Modified to Express NtHMA RNAi Polynucleotides

To determine the effect of NtHMA (660-915) RNAi polynucleotideexpression on Cd content in leaf lamina, the metal content of two (T1)transgenic lines of VA359 variety were grown in soil containing variableCd concentrations for 4 weeks. Two transgenic lines, 06T498 and 06T506,selected as kanamycin positives were screened by PCR. Several 10″ Potsfilled with sand:soil mixture were saturated with either 0, 0.1, 0.5, or5 μM CdCl₂. Three plants per treatment per transgenic line were grownfor 4 weeks by adding Hoaglands medium to the saucer. Total number ofleaves, leaf area index, leaf weight, stalk weight, and root weight wereobserved. Two middle leaves and root samples were freeze-dried and weresubjected to heavy metal analysis.

FIG. 8 shows Cd distribution between the leaf lamina and the root ofvarious second generation (T1) transgenic lines that have beengenetically modified to express NtHMA RNAi polynucleotides of interest.In FIG. 8, the Cd content of the transgenic plants was consistentlylower than that of control plants at all Cd concentrations tested (0,0.1, 0.5, and 5 μM). A reduction in Cd content of the leaf lamina (2-4.7fold) was observed in various transgenic lines tested. The Cd level forthe line 06T498 was only ˜20% of control plants at 5 μM CdCl₂. Anincrease in root Cd content (4-16 fold) was observed in varioustransgenic lines tested. The highest root Cd content (a 16 foldincrease) was observed for line 06T498 at 5 μM CdCl₂. Thus, the reducedheavy metal content in the leaf lamina/shoots in transgenic lines,expressing NtHMA (660-915) RNAi polynucleotide, suggested that thetranslocation of a substantial amount of heavy metals from the root tothe leaf lamina/shoots can be interrupted by RNAi interference. Theresults are consistent with Cd reduction observed in the leaf lamina offirst generation transgenic lines, in that the second generationtransgenic lines also demonstrated (a) reduced Cd levels in the leaflamina, and (b) increased Cd in the roots. The transgenic lines did notdemonstrate phenotypical differences in general appearance, growth, anddevelopment relative to that of control plants.

Example 10 NtHMA Polynucleotides

A NtHMA polynucleotide will generally contain phosphodiester bonds,although in some cases, nucleic acid analogs are included that may havealternate backbones, comprising, e.g., phosphoramidate,phosphorothioate, phosphorodithioate, or O-methylphophoroamiditelinkages (see Eckstein, Oligonucleotides and Analogues: A PracticalApproach, Oxford University Press); and peptide nucleic acid backbonesand linkages. Other analog nucleic acids include those with positivebackbones; non-ionic backbones, and non-ribose backbones, includingthose described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters6 and 7, ASC Symposium Series 580, Carbohydrate Modifications inAntisense Research, Sanghui & Cook, eds. Nucleic acids containing one ormore carbocyclic sugars are also included within one definition ofnucleic acids. Modifications of the ribose-phosphate backbone may bedone for a variety of reasons, e.g. to increase the stability andhalf-life of such molecules in physiological environments or as probeson a biochip. Mixtures of naturally occurring nucleic acids and analogscan be made; alternatively, mixtures of different nucleic acid analogs,and mixtures of naturally occurring nucleic acids and analogs may bemade.

A variety of references disclose such nucleic acid analogs, including,for example, phosphoramidate (Beaucage et al., Tetrahedron 49(10):1925(1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970);Sprinzl et al., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl.Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984),Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al.,Chemica Scripta 26:141 91986)), phosphorothioate (Mag et al., NucleicAcids Res. 19:1437 (1991); and U.S. Pat. No. 5,644,048),phosphorodithioate (Briu et al., J. Am. Chem. Soc. 111:2321 (1989),O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides andAnalogues: A Practical Approach, Oxford University Press), and peptidenucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc.114:1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992);Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature 380:207 (1996),all of which are incorporated by reference). Other analog nucleic acidsinclude those with positive backbones (Denpcy et al., Proc. Natl. Acad.Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Pat. Nos. 5,386,023,5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew.Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem.Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13:1597(1994); Chapters 2 and 3, ASC Symposium Series 580, “CarbohydrateModifications in Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook;Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffset al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743(1996)) and non-ribose backbones, including those described in U.S. Pat.Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series580, “Carbohydrate Modifications in Antisense Research”, Ed. Y. S.Sanghui and P. Dan Cook. Nucleic acids containing one or morecarbocyclic sugars are also included within one definition of nucleicacids (see Jenkins et al., Chem. Soc. Rev. (1995) pp 169-176). Severalnucleic acid analogs are described in Rawls, C & E News Jun. 2, 1997page 35. These references are hereby expressly incorporated byreference.

Other analogs include peptide nucleic acids (PNA) which are peptidenucleic acid analogs. These backbones are substantially non-ionic underneutral conditions, in contrast to the highly charged phosphodiesterbackbone of naturally occurring nucleic acids. This results in twoadvantages. First, the PNA backbone exhibits improved hybridizationkinetics. PNAs have larger changes in the melting temperature (Tm) formismatched versus perfectly matched basepairs. DNA and RNA typicallyexhibit a 2-4° C. drop in T_(m) for an internal mismatch. With thenon-ionic PNA backbone, the drop is closer to 7-9° C. Similarly, due totheir non-ionic nature, hybridization of the bases attached to thesebackbones is relatively insensitive to salt concentration. In addition,PNAs are not degraded by cellular enzymes, and thus can be more stable.

Among the uses of the disclosed NtHMA polynucleotides, and combinationsof fragments thereof, is the use of fragments as probes or primers or inthe development of RNAi molecules. Such fragments generally comprise atleast about 17 contiguous nucleotides of a DNA sequence. In otherembodiments, a DNA fragment comprises at least 30, or at least 60contiguous nucleotides of a DNA sequence. The basic parameters affectingthe choice of hybridization conditions and guidance for devisingsuitable conditions are set forth by Sambrook et al., 1989 and aredescribed in detail above. Using knowledge of the genetic code incombination with the amino acid sequences set forth above, sets ofdegenerate oligonucleotides can be prepared. Such oligonucleotides areuseful as primers, e.g., in polymerase chain reactions (PCR), wherebyDNA fragments are isolated and amplified. In certain embodiments,degenerate primers can be used as probes for non-human geneticlibraries. Such libraries would include but are not limited to cDNAlibraries, genomic libraries, and even electronic EST (express sequencetag) or DNA libraries. Homologous sequences identified by this methodwould then be used as probes to identify non-human homologues of theNtHMA sequence identified herein.

The disclosure also includes polynucleotides and oligonucleotides thathybridize under reduced stringency conditions, typically moderatelystringent conditions, and commonly highly stringent conditions, to anNtHMA polynucleotide described herein. The basic parameters affectingthe choice of hybridization conditions and guidance for devisingsuitable conditions are set forth by Sambrook, J., E. F. Fritsch, and T.Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11;and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al.,eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporatedherein by reference), and can be readily determined by those havingordinary skill in the art based on, for example, the length and/or basecomposition of the polynucleotide. One way of achieving moderatelystringent conditions involves the use of a prewashing solutioncontaining 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization bufferof about 50% formamide, 6×SSC, and a hybridization temperature of about55° C. (or other similar hybridization solutions, such as one containingabout 50% formamide, with a hybridization temperature of about 42° C.),and washing conditions of about 60° C., in 0.5×SSC, 0.1% SDS. Generally,highly stringent conditions are defined as hybridization conditions asabove, but with washing at approximately 68° C., 0.2×SSC, 0.1% SDS. SSPE(1×SSPE is 0.15M NaCl, 10 mM NaH₂PO4, and 1.25 mM EDTA, pH 7.4) can besubstituted for SSC (1×SSC is 0.15M NaCl and 15 mM sodium citrate) inthe hybridization and wash buffers; washes are performed for 15 minutesafter hybridization is complete. It should be understood that the washtemperature and wash salt concentration can be adjusted as necessary toachieve a desired degree of stringency by applying the basic principlesthat govern hybridization reactions and duplex stability, as known tothose skilled in the art and described further below (see, e.g.,Sambrook et al., 1989). When hybridizing a nucleic acid to a targetpolynucleotide of unknown sequence, the hybrid length is assumed to bethat of the hybridizing nucleic acid. When nucleic acids of knownsequence are hybridized, the hybrid length can be determined by aligningthe sequences of the nucleic acids and identifying the region or regionsof optimal sequence complementarity. The hybridization temperature forhybrids anticipated to be less than 50 base pairs in length should be 5to 10° C. less than the melting temperature (T_(m)) of the hybrid, whereT_(m) is determined according to the following equations. For hybridsless than 18 base pairs in length, T_(m) (° C.)=2(# of A+T bases)+4(# ofG+C bases). For hybrids above 18 base pairs in length, T_(m) (°C.)=81.5+16.6(log 10 [Na+])+0.41(% G+C)−(600/N), where N is the numberof bases in the hybrid, and [Na+] is the concentration of sodium ions inthe hybridization buffer ([Na+] for 1×SSC=0.165M). Typically, each suchhybridizing nucleic acid has a length that is at least 25% (commonly atleast 50%, 60%, or 70%, and most commonly at least 80%) of the length ofa polynucleotide of the disclosure to which it hybridizes, and has atleast 60% sequence identity (e.g., at least 70%, 75%, 80%, 85%, 90%,95%, 97.5%, or at least 99%) with a polynucleotide of the disclosure towhich it hybridizes.

Example 11 NtHMA Polypeptides

A polypeptide of the disclosure may be prepared by culturing transformedor recombinant host cells under culture conditions suitable to express apolypeptide of the disclosure. The resulting expressed polypeptide maythen be purified from such culture using known purification processes.The purification of the polypeptide may also include an affinity columncontaining agents which will bind to the polypeptide; one or more columnsteps over such affinity resins as concanavalin A-agarose,heparin-Toyopearl® or Cibacrom blue 3GA Sepharose®; one or more stepsinvolving hydrophobic interaction chromatography using such resins asphenyl ether, butyl ether, or propyl ether; or immunoaffinitychromatography. Alternatively, the polypeptide of the disclosure mayalso be expressed in a form that will facilitate purification. Forexample, it may be expressed as a fusion polypeptide, such as those ofmaltose binding polypeptide (MBP), glutathione-5-transferase (GST) orthioredoxin (TRX). Kits for expression and purification of such fusionpolypeptides are commercially available from New England BioLab(Beverly, Mass.), Pharmacia (Piscataway, N.J.), and InVitrogen,respectively. The polypeptide can also be tagged with an epitope andsubsequently purified by using a specific antibody directed to suchepitope. Finally, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify the polypeptide. Some or all of the foregoingpurification steps, in various combinations, can also be employed toprovide a substantially homogeneous recombinant polypeptide. Thepolypeptide thus purified is substantially free of other mammalianpolypeptides and is defined in accordance with the invention as an“substantially purified polypeptide”; such purified polypeptides includeNtHMA polypeptide, fragment, variant, and the like. Expression,isolation, and purification of the polypeptides and fragments of thedisclosure can be accomplished by any suitable technique, including butnot limited to the methods described herein.

It is also possible to utilize an affinity column such as a monoclonalantibody generated against polypeptides of the disclosure, toaffinity-purify expressed polypeptides. These polypeptides can beremoved from an affinity column using conventional techniques, e.g., ina high salt elution buffer and then dialyzed into a lower salt bufferfor use or by changing pH or other components depending on the affinitymatrix utilized, or be competitively removed using the naturallyoccurring substrate of the affinity moiety, such as a polypeptidederived from the disclosure.

A polypeptide of the disclosure may also be produced by knownconventional chemical synthesis. Methods for constructing thepolypeptides of the disclosure or fragments thereof by synthetic meansare known to those skilled in the art. The synthetically-constructedpolypeptide sequences, by virtue of sharing primary, secondary ortertiary structural and/or conformational characteristics with a nativepolypeptides may possess biological properties in common therewith,including biological activity.

Example 12 Anti-NtHMA Antibodies

In another embodiment, antibodies that are immunoreactive with thepolypeptides of the disclosure are provided herein. The NtHMApolypeptides, fragments, variants, fusion polypeptides, and the like, asset forth herein, can be employed as “immunogens” in producingantibodies immunoreactive therewith. Such antibodies specifically bindto the polypeptides via the antigen-binding sites of the antibody.Specifically binding antibodies are those that will specificallyrecognize and bind with NtHMA family polypeptides, homologues, andvariants, but not with other molecules. In one embodiment, theantibodies are specific for polypeptides having an NtHMA amino acidsequence of the disclosure as set forth in SEQ ID NO:2 and do notcross-react with other polypeptides.

More specifically, the polypeptides, fragment, variants, fusionpolypeptides, and the like contain antigenic determinants or epitopesthat elicit the formation of antibodies. These antigenic determinants orepitopes can be either linear or conformational (discontinuous). Linearepitopes are composed of a single section of amino acids of thepolypeptide, while conformational or discontinuous epitopes are composedof amino acids sections from different regions of the polypeptide chainthat are brought into close proximity upon polypeptide folding. Epitopescan be identified by any of the methods known in the art. Additionally,epitopes from the polypeptides of the disclosure can be used as researchreagents, in assays, and to purify specific binding antibodies fromsubstances such as polyclonal sera or supernatants from culturedhybridomas. Such epitopes or variants thereof can be produced usingtechniques known in the art such as solid-phase synthesis, chemical orenzymatic cleavage of a polypeptide, or using recombinant DNAtechnology.

Both polyclonal and monoclonal antibodies to the polypeptides of thedisclosure can be prepared by conventional techniques. See, for example,Monoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Kennet et al. (eds.), Plenum Press, New York (1980); andAntibodies: A Laboratory Manual, Harlow and Land (eds.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1988); Kohler andMilstein, (U.S. Pat. No. 4,376,110); the human B-cell hybridomatechnique (Kosbor et al., Immunology Today 4:72, 1983; Cole et al.,Proc. Natl. Acad. Sci. USA 80:2026, 1983); and the EBV-hybridomatechnique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy,Alan R. Liss, Inc., pp. 77-96). Hybridoma cell lines that producemonoclonal antibodies specific for the polypeptides of the disclosureare also contemplated herein. Such hybridomas can be produced andidentified by conventional techniques. For the production of antibodies,various host animals may be immunized by injection with an NtHMApolypeptide, fragment, variant, or mutants thereof. Such host animalsmay include, but are not limited to, rabbits, mice, and rats, to name afew. Various adjutants may be used to increase the immunologicalresponse. Depending on the host species, such adjutants include, but arenot limited to, Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjutants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Themonoclonal antibodies can be recovered by conventional techniques. Suchmonoclonal antibodies may be of any immunoglobulin class including IgG,IgM, IgE, IgA, IgD, and any subclass thereof.

The antibodies of the disclosure can also be used in assays to detectthe presence of the polypeptides or fragments of the disclosure, eitherin vitro or in vivo. The antibodies also can be employed in purifyingpolypeptides or fragments of the disclosure by immunoaffinitychromatography.

Example 13 Double-Stranded RNAs

In one embodiment, the disclosure provides double-stranded ribonucleicacid (dsRNA) molecules for inhibiting the expression of the NtHMA genein a cell (e.g., a plant cell), wherein the dsRNA comprises an antisensestrand comprising a region of complementarity which is complementary toat least a part of an mRNA formed in the expression of the NtHMA gene,and wherein the region of complementarity is less than 30 nucleotides inlength and wherein said dsRNA, upon contact with a cell expressing saidNtHMA gene, inhibits the expression of said NtHMA gene by at least 20%.The dsRNA comprises two RNA strands that are sufficiently complementaryto hybridize to form a duplex structure. One strand of the dsRNA (theantisense strand) comprises a region of complementarity that issubstantially complementary, and typically fully complementary, to atarget sequence, derived from the sequence of an mRNA formed during theexpression of the NtHMA gene, the other strand (the sense strand)comprises a region which is complementary to the antisense strand, suchthat the two strands hybridize and form a duplex structure when combinedunder suitable conditions. The duplex structure is between about 15 and30 (e.g., between about 18 and 25), typically between about 19 and 24(e.g., between 21 and 23) base pairs in length. Similarly, the region ofcomplementarity to the target sequence is between 15 and 30 (e.g.,between about 18 and 25), typically between about 19 and 24 (e.g.,between 21 and 23) base pairs in length. The dsRNA of the disclosure mayfurther comprise one or more single-stranded nucleotide overhang(s). ThedsRNA can be synthesized by standard methods known in the art as furtherdiscussed below, e.g., by use of an automated DNA synthesizer, such asare commercially available from, for example, Biosearch, AppliedBiosystems, Inc. In another aspect, an expression vector can be used toexpress an RNAi molecule in vivo.

The dsRNA of the disclosure can contain one or more mismatches to thetarget sequence. In one embodiment, the dsRNA of the disclosure containsmore than 3 mismatches. If the antisense strand of the dsRNA containsmismatches to a target sequence, it is typical that the area of mismatchnot be located in the center of the region of complementarity. If theantisense strand of the dsRNA contains mismatches to the targetsequence, it is typical that the mismatch be restricted to 5 nucleotidesfrom either end, for example 5, 4, 3, 2, or 1 nucleotide from either the5′ or 3′ end of the region of complementarity. For example, for a 23nucleotide dsRNA strand which is complementary to a region of the NtHMAgene, the dsRNA preferably does not contain any mismatch within thecentral 13 nucleotides. The methods described within the disclosure canbe used to determine whether a dsRNA containing a mismatch to a targetsequence is effective in inhibiting the expression of the NtHMA gene.

In one embodiment, at least one end of the dsRNA has a single-strandednucleotide overhang of 1 to 4 (e.g., 1 or 2 nucleotides). dsRNAs havingat least one nucleotide overhang have inhibitory properties. The dsRNAmay also have a blunt end, typically located at the 5′-end of theantisense strand.

In yet another embodiment, the dsRNA is chemically modified to enhancestability. The nucleic acids of the disclosure may be synthesized and/ormodified by methods well established in the art, such as those describedin “Current protocols in nucleic acid chemistry”, Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Chemical modifications may include,but are not limited to 2′ modifications, introduction of non-naturalbases, covalent attachment to a ligand, and replacement of phosphatelinkages with thiophosphate linkages. In this embodiment, the integrityof the duplex structure is strengthened by at least one, and typicallytwo, chemical linkages. Chemical linking may be achieved by any of avariety of well-known techniques, for example by introducing covalent,ionic or hydrogen bonds; hydrophobic interactions, van der Waals orstacking interactions; by means of metal-ion coordination, or throughuse of purine analogues.

In yet another embodiment, the nucleotides at one or both of the twosingle strands may be modified to prevent or inhibit the activation ofcellular enzymes, such as, for example, without limitation, certainnucleases. Techniques for inhibiting the activation of cellular enzymesare known in the art including, but not limited to, 2′-aminomodifications, 2′-fluoro modifications, 2′-alkyl modifications,uncharged backbone modifications, morpholino modifications, 2′-O-methylmodifications, and phosphoramidate (see, e.g., Wagner, Nat. Med. (1995)1:1116-8). Thus, at least one 2′-hydroxyl group of the nucleotides on adsRNA is replaced by a chemical group. Also, at least one nucleotide maybe modified to form a locked nucleotide. Such locked nucleotide containsa methylene or ethylene bridge that connects the 2′-oxygen of ribosewith the 4′-carbon of ribose. Oligonucleotides containing the lockednucleotide are described in Koshkin, A. A., et al., Tetrahedron (1998),54: 3607-3630) and Obika, S. et al., Tetrahedron Lett. (1998), 39:5401-5404). Introduction of a locked nucleotide into an oligonucleotideimproves the affinity for complementary sequences and increases themelting temperature by several degrees (Braasch, D. A. and D. R. Corey,Chem. Biol. (2001), 8:1-7).

Conjugating a ligand to a dsRNA can enhance its cellular absorption. Incertain instances, a hydrophobic ligand is conjugated to the dsRNA tofacilitate direct permeation of the cellular membrane. Alternatively, aligand conjugated to the dsRNA is a substrate for receptor-mediatedendocytosis. These approaches have been used to facilitate cellpermeation of antisense oligonucleotides. In certain instances,conjugation of a cationic ligand to oligonucleotides often results inimproved resistance to nucleases. Representative examples of cationicligands are propylammonium and dimethylpropylammonium. Interestingly,anti-sense oligonucleotides were reported to retain their high bindingaffinity to mRNA when the cationic ligand was dispersed throughout theoligonucleotide. See M. Manoharan Antisense & Nucleic Acid DrugDevelopment 2002, 12, 103 and references therein.

Example 15 Methods for Identifying NtHMA Modulatory Agents

The disclosure provides methods for identifying agents that can modulateNtHMA expression level and/or activity. Candidates (“a test agent”) thatmay be screened to identify NtHMA-specific modulatory activity includesmall molecules, chemicals, peptidomimetics, antibodies, peptides,polynucleotides (e.g., RNAi, siRNA, antisense or ribozyme molecules),and agents developed by computer-based design. Modulation of NtHMAincludes an increase or decrease in activity or expression. For example,a method for identifying candidates that can modulate NtHMA expressionand/or activity, comprises: contacting a sample containing an NtHMApolypeptide or polynucleotide with a test agent under conditions thatallow the test agent and the NtHMA polypeptide or polynucleotide tointeract, and measuring the expression and/or activity of the NtHMApolypeptide in the presence or absence of the test agent.

In one embodiment, a cell containing an NtHMA polynucleotide iscontacted with a test agent under conditions such that the cell and testagent are allowed to interact. Such conditions typically include normalcell culture conditions consistent with the particular cell type beingutilized, known in the art. It may be desirable to allow the test agentand the cell to interact under conditions associated with increasedtemperature or in the presence of regents that facilitate the uptake ofthe test agent by the cell. A control is treated similarly but in theabsence of the test agent. Alternatively, the NtHMA activity orexpression may be measured prior to contact with the test agent (e.g.,the standard or control measurement) and then again following contactwith the test agent. The treated cell is then compared to the controland a difference in the expression or activity of NtHMA compared to thecontrol is indicative of an agent that modulates NtHMA activity orexpression.

When NtHMA expression is being measured, detecting the amount of mRNAencoding an NtHMA polypeptide in the cell can be quantified by, forexample, PCR or Northern blot. Where a change in the amount of NtHMApolypeptide in the sample is being measured, detecting NtHMA by use ofanti-NtHMA antibodies can be used to quantify the amount of NtHMApolypeptide in the cell using known techniques. Alternatively thebiological activity (e.g., heavy metal transport) can be measured beforeand after contact with the test agent.

It will be appreciated that, although specific embodiments of theinvention have been described herein for purposes of illustration,various modifications may be made without departing from the spirit andthe scope of the invention. Accordingly, the invention is not limitedexcept as by the appended claims. Unless defined otherwise, alltechnical and scientific terms have standard meaning as commonlyunderstood to persons skilled in the art. Although exemplary methods,devices, and materials have been described with particularity,alternative methods and materials, that may be similar or equivalent tothose described herein, are applicable for making the disclosedcompositions and for practicing the disclosed methods.

Any publication cited or described herein provides relevant informationdisclosed prior to the filing date of the present application.Statements herein are not to be construed as an admission that theinventors are not entitled to antedate such disclosures.

We claim:
 1. An NtHMA RNAi construct that inhibits the expression of anNtHMA messenger RNA to which it corresponds, wherein the constructcomprises a heterologous promoter and: (a) a first sequence having atleast 95% sequence identity to a sequence selected from the groupconsisting of: exon 1 (SEQ ID NO: 5), a fragment of exon 1 (SEQ ID NO:5), exon 2 (SEQ ID NO: 7), a fragment of exon 2 (SEQ ID NO: 7), exon 3(SEQ ID NO: 9), a fragment of exon 3 (SEQ ID NO: 9), exon 4 (SEQ ID NO:11), a fragment of exon 4 (SEQ ID NO: 11), exon 5 (SEQ ID NO: 13), afragment of exon 5 (SEQ ID NO: 13), exon 6 (SEQ ID NO: 15), a fragmentof exon 6 (SEQ ID NO: 15), exon 7 (SEQ ID NO: 17), a fragment of exon 7(SEQ ID NO: 17), exon 8 (SEQ ID NO: 19), a fragment of exon 8 (SEQ IDNO: 19), exon 9 (SEQ ID NO: 21), a fragment of exon 9 (SEQ ID NO: 21),exon 10 (SEQ ID NO: 23), a fragment of exon 10 (SEQ ID NO:23), exon 11(SEQ ID NO: 25), and a fragment of exon 11 (SEQ ID NO:25); (b) a secondsequence having at least 95% sequence identity to a sequence selectedfrom the group consisting of: intron 1 (SEQ ID NO: 4), a fragment ofintron 1 (SEQ ID NO:4), intron 2 (SEQ ID NO: 6), a fragment of intron 2(SEQ ID NO:6), intron 3 (SEQ ID NO: 8), a fragment of intron 3 (SEQ IDNO:8), intron 4 (SEQ ID NO: 10), a fragment of intron 4 (SEQ ID NO: 10),intron 5 (SEQ ID NO: 12), a fragment of intron 5 (SEQ ID NO: 12), intron6 (SEQ ID NO: 14), a fragment of intron 6 (SEQ ID NO: 14), intron 7 (SEQID NO: 16), a fragment of intron 7 (SEQ ID NO: 16), intron 8 (SEQ ID NO:18), a fragment of intron 8 (SEQ ID NO: 18), intron 9 (SEQ ID NO: 20), afragment of intron 9 (SEQ ID NO: 20), intron 10 (SEQ ID NO: 22), afragment of intron 10 (SEQ ID NO: 22), intron 11 (SEQ ID NO: 24), afragment of intron 11 (SEQ ID NO: 24), intron 12 (SEQ ID NO: 26), and afragment of intron 12 (SEQ ID NO: 26); and (c) a third sequence havingat least 95% sequence identity to a sequence selected from the groupconsisting of: SEQ ID NO: 27, a fragment of SEQ ID NO: 27, SEQ ID NO:28, a fragment of SEQ ID NO: 28, SEQ ID NO: 29, a fragment of SEQ ID NO:29, SEQ ID NO: 30, a fragment of SEQ ID NO: 30, SEQ ID NO: 31, afragment of SEQ ID NO: 31, SEQ ID NO: 32, a fragment of SEQ ID NO: 32,SEQ ID NO: 33, a fragment of SEQ ID NO: 33, SEQ ID NO: 34, a fragment ofSEQ ID NO: 34, SEQ ID NO: 35, a fragment of SEQ ID NO: 35, SEQ ID NO:36, a fragment of SEQ ID NO: 36, SEQ ID NO: 37, and a fragment of SEQ IDNO: 37; wherein the third sequence is the reverse complementary sequenceof the first sequence, wherein said fragments are at least 30 contiguousnucleotides of the respective SEQ ID NO; and wherein the second sequenceis positioned between the first sequence and the third sequence, and thesecond sequence is operably-linked to the first sequence and to thethird sequence.
 2. An NtHMA RNAi construct capable of inhibiting theexpression of an NtHMA messenger RNA to which it corresponds, whereinthe construct comprises: a first sequence comprising SEQ ID NO:38, asecond sequence comprising SEQ ID NO:39, and a third sequence comprisingSEQ ID NO:40; wherein the second sequence is positioned between thefirst sequence and the third sequence, and the second sequence isoperably-linked to the first sequence and to the third sequence.
 3. AnNtHMA RNAi construct capable of inhibiting the expression of an NtHMAmessenger RNA to which it corresponds, wherein the construct comprises:a first sequence comprising SEQ ID NO:42, a second sequence comprisingSEQ ID NO:43, and a third sequence comprising SEQ ID NO:44; wherein thesecond sequence is positioned between the first sequence and the thirdsequence, and the second sequence is operably-linked to the firstsequence and to the third sequence.
 4. An NtHMA RNAi construct thatinhibits the expression of an NtHMA messenger RNA to which itcorresponds, wherein the construct comprises a heterologous promoterand: (a) and (c); or (a) and (b) and (c), wherein (a) is a firstsequence having at least 95% sequence identity to a sequence selectedfrom the group consisting of: exon 1 (SEQ ID NO: 5), a fragment of exon1 (SEQ ID NO: 5), exon 2 (SEQ ID NO: 7), a fragment of exon 2 (SEQ IDNO: 7), exon 3 (SEQ ID NO: 9), a fragment of exon 3 (SEQ ID NO: 9), exon4 (SEQ ID NO: 11), a fragment of exon 4 (SEQ ID NO: 11), exon 5 (SEQ IDNO: 13), a fragment of exon 5 (SEQ ID NO:13), exon 6 (SEQ. ID NO:15), afragment of exon 6 (SEQ ID NO:15), exon 7 (SEQ ID NO:17), a fragment ofexon 7 (SEQ ID NO:17), exon 8 (SEQ ID NO:19), a fragment of exon 8 (SEQID NO:19), exon 9 (SEQ ID NO:2), a fragment of exon 9 (SEQ ID NO:21),exon 10 (SEQ ID NO:23), a fragment of exon 10 (SEQ ID NO:23), exon 11(SEQ ID NO:25), and a fragment of exon 11 (SEQ ID NO:25); wherein (b) isa second sequence having at least 95% sequence identity to a sequenceselected from the group consisting of: intron 1 (SEQ ID NO: 4), afragment of intron 1 (SEQ ID NO: 4), intron 2 (SEQ ID NO: 6), a fragmentof intron 2 (SEQ ID NO:6), intron 3 (SEQ ID NO: 8), a fragment of intron3 (SEQ ID NO: 8), intron 4 (SEQ ID NO: 10), a fragment of intron 4 (SEQID NO: 10), intron 5 (SEQ ID NO:12), a fragment of intron 5 (SEQ ID NO:12), intron 6 (SEQ ID NO:14), a fragment of intron 6 (SEQ ID NO: 14),intron 7 (SEQ ID NO:16), a fragment of intron 7 (SEQ ID NO:16), intron 8(SEQ ID NO: 18), a fragment of intron 8 (SEQ ID NO: 18), intron 9 (SEQID NO: 20), a fragment of intron 9 (SEQ ID NO:20), intron 10 (SEQ ID NO:22), a fragment of intron 10 (SEQ ID NO:22), intron 11 (SEQ ID NO: 24),a fragment of intron 11 (SEQ ID NO: 24), intron 12 (SEQ ID NO: 26), anda fragment of intron 12 (SEQ ID NO: 26); and wherein (c) is a thirdsequence having at least 95% sequence identity to a sequence selectedfrom the group consisting of: SEQ ID NO: 27, a fragment of SEQ ID NO:27, SEQ ID NO: 28, a fragment of SEQ ID NO: 28, SEQ ID NO: 29, afragment of SEQ ID NO: 29, SEQ ID NO: 30, a fragment of SEQ ID NO: 30,SEQ ID NO: 31, a fragment of SEQ ID NO: 31, SEQ ID NO: 32, a fragment ofSEQ ID NO: 32, SEQ ID NO: 33, a fragment of SEQ ID NO: 33, SEQ ID NO:34, a fragment of SEQ ID NO: 34, SEQ ID NO: 35, a fragment of SEQ ID NO:35, SEQ ID NO: 36, a fragment of SEQ ID NO: 36, SEQ ID NO: 37, and afragment of SEQ ID NO: 37; wherein the third sequence is the reversecomplementary sequence of the first sequence, wherein said fragments areat least 30 contiguous nucleotides of the respective SEQ ID NO; andfurther wherein when said construct comprises (a) and (b) and (c), thesecond sequence is positioned between the first sequence and the thirdsequence, and the second sequence Is operably-linked to the firstsequence and to the third sequence.